Relationship between oxidative stress levels and activation state on a hepatic stellate cell line.

BACKGROUND/AIMS: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast-like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype. METHODS/RESULTS: We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2',7'-dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N-acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status. CONCLUSIONS: The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype.

Hepatic stellate cells are intralobular connective tissue cells expressing the myofibroblast or the lipocyte phenotypes. They participate in homeostasis of the liver extracellular matrix, repair, regeneration, and fibrosis under the former phenotype, and control the retinol metabolism, storage, and release under the latter one. They are heterogeneous in terms of their tissue distribution, function, and expression of cytoskeletal proteins. We have studied the expressions of intermediate filaments in the cloned GRX cell line representative of murine hepatic stellate cells, by immunolabeling, reverse transcription polymerase chain reaction (RT-PCR), immunoprecipitation and Western blots. GRX cells expressed vimentin, desmin, glial fibrillary acidic protein (GFAP), and smooth muscle alpha actin (SM-alphaA). Vimentin, desmin, and SMN-alphaA were expressed in all cultures. GFAP showed a heterogeneous intensity of expression and did not form a filamentous cytoskeletal network, showing a distinct punctuate cytoplasmic distribution. When activated by inflammatory mediators, GRX cells increased expression of desmin and GFAP. Retinol-mediated induction of the lipocyte phenotype elicited a strong decrease of intermediate filament protein expression and the collapse of the filamentous structure of the cytoskeleton. Quiescent hepatic stellate precursors can respond to physiologic or pathologic stimuli, expressing activated myofibroblast or lipocyte phenotypes with distinct patterns of cytoskeleton structure, metabolic function, and interaction with the tissue environment.

Lipid metabolism during in vitro induction of the lipocyte phenotype in hepatic stellate cells.

Molecular mechanisms of lipid synthesis and their controls in hepatic stellate cells are not known. We have previously proposed that, in contrast to other fat storing cells, hepatic stellate cells are not involved in energy storage, but they represent a particular cell population specialized in storage of lipid-soluble substances, the major one being probably retinol. In agreement with this hypothesis, induction of the lipocyte phenotype in stellate cells is not under the control of insulin, but responds to retinoids and other molecules that modify the gene expression program in these cells. In the present study we have monitored the activity of the two major enzymes involved in lipid synthesis during the induction of the lipocyte phenotype in hepatic stellate cells: glycerol-3-phosphate dehydrogenase (GPDH) that mediates the de novo lipid synthesis, and lipoprotein lipase that mediates incorporation of plasma lipids. In early stages of lipocyte induction, both pathways of lipid synthesis are activated. When lipocytes have already constituted the lipid droplets, lipoprotein lipase pathway is downregulated, while GPDH activity remains high. Adult liver has been reported to lack lipoprotein lipase, but under stress, lipase activity was detected around and at the surface of the intrahepatic vasculature. We have now shown that the lipase activity can be induced in the hepatic stellate cells, located in the Disse's space. The high lipoprotein lipase activity under acute induction of lipocyte phenotype, followed by the low activity under conditions of metabolic equilibrium, are in compass with the increased activity of this enzyme under stress, and its low activity in adult liver parenchyma under normal conditions.

Phospholipid modifications during conversion of hepatic myofibroblasts into lipocytes (Ito-cells).

Connective tissue cells of liver parenchyma (perisinusoidal myofibroblasts) can be induced to express the lipocyte (Ito cell) phenotype. We have studied phospholipid synthesis and phosphate incorporation during this in vitro conversion, induced by insulin and/or indomethacin, in the established murine cell line GRX. Phospholipid synthesis, measured by [14C]acetate incorporation, was increased after a full induction of the lipocyte phenotype. The 32Pi incorporation into phospholipids was increased from the beginning of induction. Phosphatidic acid and phosphatidylinositol synthesis were increased early in the induction, whilst the increase of major constitutive phospholipids was significant only after the full lipocyte phenotype induction. The presence of unsaturated fatty acids in phospholipids was increased in lipocytes. Linoleic acid was present only in diacylglycerols and in phosphatidylinositol. Since we have shown previously that linoleic acid was not present in triacylglycerols, this result indicates the importance of future studies on activation of phosphatidylinositol cycles in induction of lipocyte phenotype in liver connective tissue cells.

Neutral lipid synthesis and accumulation during in vitro induction of the lipocyte phenotype in hepatic connective tissue cells.

Connective tissue cells of liver parenchyma are known as hepatic myofibroblasts and lipocytes (fat-storing cells, Ito-cells). They are considered to belong to a single cell lineage, that may switch between these two phenotypes. We have studied cellular and molecular parameters and controls of this switch in the murine GRX cell line, established from liver fibro-granulomatous lesions induced by schistosomal infection. Accumulation of neutral lipids (triacylglycerols, monoalkyl-diacylglycerol, cholesterol) was monitored. It was dependent upon induction with indomethacin. Insulin alone did not induce lipid accumulation in GRX cells, but in cells induced by indomethacin it increased the quantity of stored lipids. We propose that hepatic lipocytes are not cells directly involved in energy storage, but that they represent a particular cell population specialized in storage and in controls of the homoeostasis of lipid-soluble substances at the systemic level.

Glycoprotein biosynthesis by testes of 40-day-old rats subjected to protein malnutrition.

The testes of 40-day-old rats subjected to protein malnutrition show a marked delay in maturation of the seminiferous epithelium, as well as greater mannose incorporation into glycoprotein than observed in normal animals of the same age. Testes were incubated for 1 h with [2-3H]mannose and germ cells were then separated by the Staput method. Mannose incorporation occurred in the same cell fraction, i.e. the spermatocytes, both in normally fed and protein-undernourished animals. These data were confirmed by incubating the cells previously isolated on the gradient with [2-3H]mannose. Comparison of these data with results obtained in previous studies on 20-day-old animals in which mannose incorporation was lower in undernourished rats suggests that the differences observed in the present study between the experimental groups are due to alterations in the germ cells.

In vitro induction of the fat-storing phenotype in a liver connective tissue cell line-GRX.

Liver connective tissue cells have been characterized as perisinusoidal myofibroblasts and hepatic lipocytes (Ito cells, fat-storing cells). A concept of a single mesenchymal cell population that may be modulated between these two phenotypes has been postulated. We have previously established a continuous murine cell line, GRX, obtained from fibrotic granulomatous lesions induced by schistosomal infection in mouse liver. This cell line is considered to represent liver myofibroblasts. In the present study we have induced the conversion of these cells into lipocyte (fat storing) phenotype by treatment with insulin and indomethacin. We have quantified the lipid synthesis and the increase of activity of involved enzymes during the induction of the fat-storing phenotype and described modifications of cell organization along this modulation of cell functions.

[Paroxysmal tachycardia with long QRS (left branch block type) in a female patient with accessory nodo-ventricular pathway. Role of the anomalous pathway in the electrogenesis of the arrhythmia]. / Tachicardia parossistica a QRS larghi (tipo blocco di branca sinistra) in una paziente con via accessoria nodo-ventricolare. Ruolo della via anomala nella elettrogenesi dell'aritmia.

The authors report a case of paroxysmal tachycardia with widened QRS (left bundle branch block morphology), where the electrophysiologic investigation shows an accessory nodo-ventricular pathway and suggests its involvement, as descendent (anterograde) pathway in a circular movement, the ascendant (retrograde) limb of which is the nodo-hisian pathway.

[Ventricular fibrillation induced by transesophageal stimulation performed for the treatment of atrial flutter]. / Fibrillazione ventricolare indotta dalla stimolazione transesofagea attuata per il trattamento di un flutter atriale.

The authors describe a case of ventricular fibrillation occasionally induced by high rate transesophageal pacing, performed to treat an atrial flutter. They conclude that, although this technique is generally safe and well tolerated, it must be performed exclusively where an intensive care can be provided.

Effect of protein malnutrition on glycoprotein synthesis by testes of 20-day-old rats.

The effects of protein malnutrition during the nursing period on glycoprotein biosynthesis by testes of 20-day-old rats was studied. Pregnant Wistar rats were housed individually. On the day of delivery they were divided into two groups: one was fed a control diet (25% casein) and the other a low-protein diet (8% casein) for a period of 20 days. Body and testis weights of pups suckled by the malnourished mothers were significantly lower than those of the pups suckled by normally-fed mothers. The seminiferous tubules o malnourished rats showed a significant decrease in diameter and in the stage of development of spermatogenesis. Whole testes of normally-fed 20-day-old rats showed significantly greater [2-3H]mannose incorporation into glycoproteins than did the testes of malnourished rats of the same age. The microsomes of normally-fed rats showed significantly higher GDP: mannose polyprenyl mannosyl transferase activity than did microsomes from malnourished rats, and this difference increased when exogenous dolichyl-phosphate was added to the incubation medium. These results indicate that protein malnutrition decreases GDP: mannose polyprenyl mannosyl transferase activity in the microsomes of testes from 20-day-old rats.

[Plasma levels of anti-arrhythmic drugs and clinical effectiveness in the treatment of ventricular arrhythmias]. / Livelli plasmatici dei farmaci antiaritmici ed efficacia clinica nel trattamento delle aritmie ventricolari.

In spite of the ratio between plasmatic concentration and antiarrhythmic effects of a drug many factors can be influenced by many factors, the plasma level monitoring plays an important clinical role. The efficacy of the antiarrhythmic therapy should be evaluated not only by means of ECG-Holter monitoring and/or electrophysiological study but also by the means of determination of plasmatic and tissular concentration of a drug and metabolites. This latter is indispensable in the evaluation of new antiarrhythmic drugs and of a dose/effect relationship during acute and chronic therapy.

[Hemodynamic aspects of torsade de pointes: echocardiographic study of a case]. / Aspetti emodinamici della torsione di punta: studio ecocardiografico di un caso.

In a patient with complete heart block complicated by "Torsade de point" (T.D.P.) we were able to record an M-mode echocardiogram during an attack of this peculiar ventricular tachyarrhythmia. The aortic valve opening was inconstant, incomplete and unequal during the T.D.P. (13 sec.), although the rate of the tachyarrhythmia was almost constant. On the other hand, during a subsequent ventricular pacing at a comparable rate like, the aortic valve opening was constant and complete. On the basis of these observations we conclude that the reduction of stroke volume, observed in T.D.P., is mainly due to mechanical failure as a result of partial desynchronization of the ventricular activation, as in ventricular fibrillation in which, however, the desynchronization is complete.

[Multiform ventricular tachycardias and Torsades de Pointe]. / Tachicardie ventricolari polimorfe e torsioni di punta.

The term Torsade de Pointe (T.d.P.) was first introduced by Dessertenne (1966) to designate an unusual ventricular tachyarrhythmia characterized by paroxysms of V.T. at rates typically greater than 200 beats/m, in which the QRS morphology shows alternating polarity in a modulating pattern, so that the complexes appear to be twisting around the baseline. This arrhythmia, triggered by E.V.B. falling in the vulnerable period of a previous beat, occurs in the setting of a prolonged Q-T interval, is generally self-limiting and, occasionally, degenerates into V.F. Similar morphologic features may be observed in patients with a normal Q-T interval. The duration of Q-T interval has important therapeutic implications. The arrhythmia occurring in the setting of a prolonged Q-T requires strict avoidance of all drugs that may potentially further delay repolarization (including class I antiarrhythmic agents), whereas that occurring with a normal Q-T interval usually responds to conventional therapy (including administration of class I antiarrhythmic agents). Thus, it seems reasonable to reserve the term T.d.P. exclusively to the forms with prolonged Q-T interval and to define those with normal Q-T interval with the name of multiform (or polimorphous) ventricular tachycardia (M.V.T.). In this paper the Authors discuss the most important electrocardiographic, clinical, aetiological, electrogenetic features of both the T.d.P. and M.V.T., on the basis of their own experience.

Formation of lipid-linked sugars in mycelial and yeast-like forms of Mucor rouxii.

Cell wall fragments from both yeast-like and mycelial forms of the dimorphic fungus Mucor rouxii were used as enzymatic preparations to study the synthesis and role of prenyl-phospho-sugars in these systems. In the presence of GDP [14C] mannose two main products were formed. One of them was characterized as dolichol-monophosphate beta-mannose on the following basis: solubility in organic solvents, behaviour upon paper chromatography, DEAE cellulose column chromatography, mild acid hydrolysis, alkali treatment, catalytic reduction and phenol degradation. The other product was identified as a glycoprotein containing a single mannose unit linked to a serine or threonine residue. It was degraded with pronase and by mild NaOH-NaBH4 treatment all the radioactivity was released as free mannitol. When UDP [14C] glucose was employed as sugar donor two butanol soluble components were isolated. One of them (25%) was characterized as dolichol-monophosphate-beta-glucose on the basis of the same criteria as described above. The other one (75%) was neutral and was not studied in detail. Mycelial enzymes were about 40 times more active in the synthesis of the dolichol derivatives. In addition, large amounts of glycogen were detected. The role that both dolichol derivatives might play in glycoprotein biosynthesis is discussed.

[Drug treatment of a trial fibrillation in patients with Wolff-Parkinson-White syndrome]. / Esperienze di trattamento farmacologico della fibrillazione atriale in pazienti con sindrome di Wolff - Parkinson - White.

In 10 patients with ventricular preexcitation (Kent bundle), in whom atrial fibrillation (A.F.) was present, the effect of some common antiarrhythmic drugs on the conduction through the anomalous pathway, and on the ventricular rate was estimated. Procainamide caused transient complete block in the accessory pathway (disappearance of the aberrant QRS) and marked reduction of the average ventricular rate in all instances. Lidocaine caused incomplete block in the accessory pathway (reduction of the number of the aberrant QRS) and significant reduction of the average ventricular rate in all tested subjects. Amiodarone slowed the ventricular rate (increase of the average and minimum R-R intervals between wide QRS complexes) in two patients, but it did not block the anomalous pathway (all QRS complexes remained aberrant); whereas in 1 patient the ventricular rate became faster and regular and the patient had syncope, while the QRS remained always aberrant. This response was probably due to the change of A.F. into atrial flutter with atrio-ventricular conduction through the anomalous pathway only. Digitalis increased the average ventricular rate and shortened the minimum R-R interval between aberrant QRS complexes 3 out of 3 times. On the basis of our experience and of the data in the literature, we conclude that, in the management of A.F. in patients with W.P.W. syndrome:--the most effective drugs are those of the 1st group of Singh and Hauswirth classification (especially Procainamide and Ajmaline);--Lidocaine is less effective, but not ineffective and its utilization may be recommended whenever the previous drugs may be hazardous;--Amiodarone, although capable of modifying the electrophysiologic properties both of the anomalous pathway and of the A-V node, seems to be less reliable;--the drugs which influence only the A-V node (Verapamil, beta-Blockers, etc.) are quite ineffective;--finally, the Digitalis is not suitable because this drug increases the ventricular rate by decreasing the effective refractory period (ERP) of the anomalous pathway.

[Electrophysiologic demonstration of dual atrioventricular nodal pathways and final common pathway in a case of A-V nodal paroxysmal tachycardia. Considerations on the functional complexity of the A-V node (author's transl)]. / Dimostrazione elettrofisiologica di doppia via nodale e di una via finale comune in un caso di tachicardia parossistica da rientro nel nodo A-V.

The AA. studied the A-V nodal conduction using the technique of induced PAB in a patient with A-V reentrant paroxysmal tachycardia. They observed that the conduction through the A-V node failed when coupling intervals A1-A2 were between 280 and 260 msec and, after, recovered, with consistent slackening, when A1-A2 intervals were shortened, until the atrial ERP was reached. This uncommon response indicates the functional complexity of the A-V node and, particularly, suggests the presence of a final common pathway distal to the fast and slow A-V pathways, that are the anatomic-functional basis of the reentry circuit in A-V nodal paroxismal tachycardia.