Anne Marie Jezequel

Regulation of extracellular matrix synthesis by transforming growth factor β1 in human fat-storing cells

BACKGROUND Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

A morphometric study of the epithelium lining the rat intrahepatic biliary tree

BACKGROUND/AIMS Morphological and functional heterogeneity of intrahepatic bile duct epithelial cells has been suggested in situ and in isolated cholangiocytes. The aim of this study was to evaluate if: (a) bile ducts, when isolated, maintain morphometric parameters similar to ducts in situ, (b) cellular organelles show heterogeneity in ducts of different size, and (c) some features permit different classes of bile ducts to be distinguished. METHODS Studies in situ were conducted on normal liver processed for light or electron microscopy. Data were also obtained from preparations of intrahepatic biliary tree isolated from rat liver. The whole biliary tree was cut at different levels to obtain bile ducts of different diameter. The diameter of ducts, the number of lining cells, the size and the area of individual cells, the nucleo/cytoplasmic ratio, the volume density of mitochondria, endoplasmic reticulum, Golgi complex and lysosomes have been evaluated. RESULTS The diameter of intrahepatic bile ducts ranged from 5 to 100 micrograms and the area of lining cells ranged from 8 to 100 micrograms2. A highly significant linear relationship existed between duct diameter and bile duct epithelial cell area (r = 0.97, p < 0.001) or number of lining cells (r = 0.96, p < 0.001). The volume density of mitochondria ranged from 7.58 +/- 2.0% of cytoplasmic volume in the smallest isolated bile ducts to 8.50 +/- 2.7% in the largest (p = NS). The volume density of lysosomes was low and was not significantly different in ducts of different size. Rough endoplasmic reticulum was inconspicuous in the smallest ducts and increased only slightly in the largest. The inverse relationship between the nucleo/cytoplasmic ratio and duct diameter was striking (r = -0.78, p < 0.001). All morphometric data were equivalent if bile ducts were evaluated in situ or in isolated fragments. Taken together, the data allowed bile ducts to be classified into 3 classes: < 10, 10-50, and > 50 micrograms in diameter. DISCUSSION Our data show that (a) isolated bile ducts maintain morphometric characteristics similar to the tissue in situ, (b) a low grade of morphological heterogeneity is evident for intracellular organelles in ducts of different diameter and (c) the diameter of ducts, the number of lining cells and especially the nucleo/cytoplasmic ratio may indicate the origin of fragments examined where functional studies are being considered.

Intracellular pathways mediating Na+/H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells

BACKGROUND & AIMS The Na+/H+ exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na+/H+ exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na+/H+ exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na+/H+ exchanger by PDGF in HSCs. METHODS The activity of the Na+/H+ exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na+/H+ exchange protein expression was evaluated by means of Western blot. RESULTS PDGF induced a significant increase in the activity of the Na+/H+ exchanger without modifying protein expression. Inhibition of the calcium/calmodulin- and protein kinase C-dependent pathways resulted in a significant inhibition of both Na+/H+ exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na+/H+ exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na+/H+ exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase- and of phosphatidylinositol 3-kinase-dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na+/H+ exchanger. CONCLUSIONS Activation of the Na+/H+ exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C-dependent pathways. PDGF-induced HSC proliferation is mediated by Na+/H+ exchange-dependent and -independent pathways.

An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver

The main pathological feature of liver fibrosis is the accumulation of extracellular matrix associated with hyperplasia and activation of perisinusoidal (Ito) cells (PSC) to myofibroblast-like cells. Interleukin-1 enhances collagen synthesis by increasing the proliferative activity of cultured PSC and has been implicated in the pathogenesis of hepatic fibrosis.Interleukin-1 receptor antagonist (IL-1ra) can block the binding of IL-1 to its receptors and act as a natural inhibitor of IL-1. We have examined whether the administration of IL-1ra can interfere with the development of experimental cirrhosis induced by dimethylnitrosamine (DMN). Rats were divided in three groups and received respectively DMN, DMN+IL-1ra and IL-1ra. For each group the collagen content of the hepatic tissue and the volume density of the inflammatory infiltrate were measured. Immunostaining for laminin and alpha-smooth muscle actin were also performed.In animals given DMN+IL-1ra we observed a decreased deposition of laminin and collagen, and a decreased number of laminin-positive PSC and of alpha-smooth muscle actin reactive cells, compared with animals receiving DMN alone. The present findings suggest that the early activation of PSC in vivo is at least in part mediated by IL-1 and confirm that the administration of IL-1ra may be of interest in modifying the biological effects of IL-1.

Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-induced cirrhosis: An immunohistochemical study

Proliferating lipocytes (fat-storing cells or perisinusoidal stellate cells of the liver) were detected by in vivo incorporation of bromodeoxyuridine (BrdU) in an experimental model of cirrhosis in the rat by dimethylnitrosamine. Lipocytes were identified by sequential double immunohistochemical staining on frozen sections using anti-desmin antibodies as a marker of cytoplasmic intermediate filaments followed by anti-BrdU antibodies to identify S-phase nuclei in animals treated for 7, 14 or 21 days. The number of desmin-positive (lipocytes) and desmin-negative (Kupffer and endothelial cells) sinusoidal cells incorporating BrdU was recorded. The labelling index of lipocytes was calculated as the percentage of BrdU-labelled desmin-positive cells with respect to total number of lipocytes. In control animals, when the total number of lipocytes was 153.9 +/- 11/mm2 (mean +/- 1 S.E.) the number of desmin-positive S-phase sinusoidal cells never exceeded 6.8 +/- 1.2/mm2 with a maximum labelling index of 4.3 +/- 0.5%. At 7 days of treatment, the values were respectively 236 +/- 26.5/mm2, 53.2 +/- 5.9/mm2 and 22.6 +/- 0.5% (p less than 0.001 vs. controls), while, at 21 days they were 272.5 +/- 21.2/mm2, 23.3 +/- 4.0/mm2 and 8.5 +/- 1.1% respectively (p less than 0.01). These results show that hyperplasia of lipocytes represents an early reaction to dimethylnitrosamine-induced liver injury. The local accumulation of lipocytes appears to occur in areas where fibrous septa develop later on.

Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor, insulin-like growth factor 1 and insulin

BACKGROUND/AIMS The Na+/H+ exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na+/H+ exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin. METHODS/RESULTS pH(i) regulation was mainly dependent on the activity of the Na+/H+ exchanger, which was evaluated by measuring pH(i) recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na+/H+ exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na+/H+ exchanger. Amiloride (100 micromol/l) and 20 micromol/l 5-N-ethyl-N-isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na+/Ca2+ exchanger. CONCLUSIONS The Na+/H+ exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na+/H+ exchange-independent.

Quantitative study of apoptosis in normal rat gastroduodenal mucosa

The occurrence of apoptosis in the normal gastrointestinal mucosa has been given little consideration until now, although the phenomenon may be of interest in the light of recent hypotheses about its role in physiological cell renewal. In the present study, a quantitative evaluation conducted on normal gastric and duodenal mucosa of young rats has shown that apoptosis is a rare but constant phenomenon: 1.4±1.1 (mean±1 s.d.) apoptotic bodies were observed within the surface epithelium of single gastric pits and 3±1 in duodenal villi. In both situations, the apoptosis showed a preferential localization in the juxtaluminal segments of the epithelium. This phenomenon appears distinct from passive exfoliation of mucosal cells and, as an expression of ‘programmed cell death’, it is likely to contribute to the normal intestinal epithelial cell turnover.

Enumeration of S-phase cells in normal rat liver by immunohistochemistry using bromodeoxyuridine-antibromodeoxyuridine system

The visualization of incorporation sites of the thymidine analog bromodeoxyuridine (BrdU) into DNA, detected by immunocytochemistry, has been proposed as an index of the percentage of S-phase cells in a variety of tissues and as an easy, less expensive alternative to autoradiography. This technique has not yet been applied to the study of physiological cell renewal in the normal liver. In the present study, results obtained with this method in the liver of normal young adult rats is reported. BrdU was administeredin vivo and subsequent incorporation was detected by the PAP technique using monoclonal anti-BrdU antibodies. The nuclei exhibiting a positive reaction within the liver were few and accounted for about 0.45% of all hepatocytes. Positive cells were located preferentially in zone 1, which contained 82.7% of the labeled cells. Zone 2 contained 15.4%, while only 1.9% of the labeled cells were found in zone 3. Positive-staining Kupffer cell nuclei were rare (about 0.5% of all Kupffer cells) and were distributed randomly in the hepatic lobule. These findings provide quantitative data about hepatocyte renewal in the normal liver in the absence of a growth stimulus. The simplicity and the reproducibility of this technique suggests that further application of this method in situations assessing hepatic regeneration are indicated.

Age and sex related changes of plasma membrane fluidity in isolated rat hepatocytes

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.

Rearrangement of the cytoskeletal network induced by platelet-derived growth factor in rat hepatic stellate cells: role of different intracellular signalling pathways.

BACKGROUND/AIMS Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC. METHODS/RESULTS Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C. CONCLUSIONS PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.