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A Pietrangelo

Publications and source records attributed to A Pietrangelo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Diacerhein blocks iron regulatory protein activation in inflamed human monocytes.

Iron Regulatory Proteins (IRPs), by modulating expression of ferritin, which stores excess iron in a non toxic form, and transferrin receptor, which controls iron uptake, are the main controller of cellular iron metabolism. During inflammation, modification of IRP activity may affect iron availability, free radical generation and cytokine gene response in inflammatory cells. In the present study we tested the effect of inflammatory stimuli on IRP function in a human monocytic-macrophagic cell line and the possibility of interfering with these pathways by using an antiinflammatory compound, diacerhein (DAR). IRP activity was enhanced by interferon gamma/lipopolysaccarhide (IFN/LPS), and this effect was consistently counteracted by increasing concentrations of DAR. No direct effect of DAR on IRP activity was found in vitro. However, in vivo, similar IRP activation was achieved by exposing cells to nitric oxide (NO) donors and the LPS/IFN-induced activation of IRP was reversed by NO inhibitors. Interestingly, NO-induced IRP activation was efficiently blocked by DAR. These data show for the first time that a clinically useful antiinflammatory compound, DAR, interferes with IRP activation by NO in inflammed human cells.

Anthraquinones↗
PubMed1998

Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis.

In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis. To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by light microscopy. Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs. However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.

Animals↗
PubMed1998

Molecular genetics and control of iron metabolism in hemochromatosis.

BACKGROUND AND OBJECTIVES: Hereditary hemochromatosis (HC) is an inborn error of iron metabolism that leads to progressive iron overload. Considerable advances in the knowledge of molecular events in iron metabolism have been recently obtained. These molecular findings, the cloning of the gene responsible for HC (HFE gene) and the results of preliminary studies on the HFE protein prompted us to review this topic. INFORMATION SOURCES: The material examined in this review article includes papers and abstracts published in the Journals covered by the Science Citation Index and Medline. The authors have been working in the field of HC for several years and have contributed eleven of the quoted papers. STATE OF ART AND PERSPECTIVE: HC is now recognized as the genetic disease characterized by the homozygosity for the Cys-->Tyr substitution at position 282 (C282Y) of the HFE protein. The mutation abolishes the association of the HFE protein with beta 2-microglobulin (beta 2M), making the complex unable to gain the cell surface. Thus HC is an example of abnormal trafficking of the corresponding proteins. It is clear by the analysis of its structure that HFE protein is not an iron transporter itself, but has a regulatory role in iron metabolism. Its peculiar localization in the crypt cells of the small intestine suggests an important role in iron trafficking at this level. However, other proteins are involved in iron uptake, as the recently cloned Nramp2, the first iron transporter discovered in mammalians. Nramp2 has a recognized role both in the intestinal iron uptake and in the iron transport within the erythroblast. The relationships between HFE and Nramp2 are still unexplored. The recent association of HFE gene with transferrin receptor (TfR) in trophoblast cells opens new possibilities on its role in cellular iron uptake. The existence of other forms of genetic iron overload suggests that the scenario of iron proteins is not yet fully defined. Further studies in this field will contribute to our knowledge of iron metabolism regulation in humans.

Hemochromatosis↗
PubMed1997

Inappropriately high iron regulatory protein activity in monocytes of patients with genetic hemochromatosis.

In genetic hemochromatosis (GH), excess iron is deposited in parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells until the later stages of the disease. As iron absorption is inversely related to RE cells stores, a failure of RE to retain iron has been proposed as the basic defect in GH. In RE cells of GH subjects, we examined the activity of iron regulatory protein (IRP), a reliable indicator of the elusive regulatory labile iron pool, which modulates cellular iron homeostasis through control of ferritin (Ft) and transferrin receptor gene expression. RNA-bandshift assays showed a significant increase in IRP activity in monocytes from 16 patients with untreated GH compared with 28 control subjects (1.5-fold) and five patients with secondary hemochromatosis (SH) with similar iron burden (fourfold). In 17 phlebotomy-treated GH patients, IRP activity did not differ from that of control subjects. In both GH and SH monocyte-macrophages, Ft content increased by twofold and the L subunit-rich isoferritin profile was unchanged as compared with controls. IRP activity was still upregulated in vitro in monocyte-derived macrophages of GH subjects but, following manipulations of iron levels, was modulated normally. Therefore, the sustained activity of monocyte IRP found in vivo in monocytes of GH patients is not due to an inherent defect of its control, but is rather the expression of a critical abnormality of iron metabolism, eg, a paradoxical contraction of the regulatory iron pool. By preventing Ft mRNA translation, high IRP activity in monocytes may represent a molecular mechanism contributing to the inadequate Ft accumulation and insufficient RE iron storage in GH.

Adult↗
PubMed1996

C/EBP-beta/LAP controls down-regulation of albumin gene transcription during liver regeneration.

Expression of the albumin gene in the liver is controlled by several liver-enriched transcription factors. However, the mechanisms which contribute to its regulation during pathophysiological states, such as liver regeneration, are still little understood. In the present study we found that during liver regeneration down-regulation of albumin mRNA expression is transcriptionally controlled through a minimal element (nucleotide -170 to +22) of the albumin promoter and is observed mainly during the G1 phase of the cell cycle, while high levels of albumin expression are preserved at later time points. Decreased albumin mRNA levels correlate with a dramatic increase in nuclear expression of C/EBP-beta/LAP, a protein known to bind to the D site of the albumin promoter and also to be involved in cell cycle control. In contrast, nuclear expression of other factors such as HNF-1 or C/EBP-alpha, which also have been shown to transcriptionally control albumin expression, is either unchanged or slightly decreased. We show that pre- and post-translational mechanisms are involved in the higher nuclear expression of C/EBP-beta/LAP as early as 1 h after hepatectomy, which also leads to its increased binding toward the D site of the albumin promoter. Finally, in vitro transcription assays with liver nuclear extracts and recombinant C/EBP-beta/LAP demonstrate that C/EBP-beta/LAP can directly down-regulate transcription mediated by the minimal element of the albumin promoter. Additionally the inhibitory role of C/EBP-beta/LAP on the albumin minimal promoter could be confirmed by transfection experiments in hepatoma cells. These results indicate that C/EBP-beta/LAP, while enhancing transcription of cell cycle-related genes and controlling G1/S phase checkpoint, down-regulates a major liver function, i.e. albumin synthesis, to prepare the hepatocyte for entry into the cell cycle.

Albumins↗
PubMed1996

Expression of messenger RNA for liver functions following 70% and 90% hepatectomy.

AIMS/METHODS: The effect of moderate and severe reduction of the functional liver mass on gene expression for liver functions was studied in rats following 70% and 90% hepatectomy. At intervals up to 24 h after operation rats were killed and RNA was extracted from the remaining liver tissue. By slot-blot hybridization mRNA steady-state levels were determined for enzymes involved in metabolic 'liver-specific' functions, acute phase proteins, 'house-keeping', and growth-related proteins. Results were expressed as per cent of levels in a pool from fed control rats of the same gender and age. RESULTS: Among 'liver-specific' metabolic functions only expression of gluconeogenesis, represented by phosphoenol carboxykinase mRNA, was augmented initially, followed by a fall to very low values after 90% hepatectomy. The drug metabolizing system represented by CYP2B1/2 mRNA was reduced to half of the control values. Expression of urea synthesis, as reflected by carbamoylphosphate synthetase mRNA, showed a gradual decline after 90% hepatectomy, in contrast to rising levels of argininosuccinate lyase and arginase mRNA, possibly serving polyamine rather than urea synthesis. The mRNA level of the acute phase protein alpha 1-acid glycoprotein showed a smaller and later rise in 90% than in 70% hepatectomized rats, whereas that of alpha 2-macroglobulin only increased after 90% hepatectomy like the 'house-keeping' beta-actin mRNA. A rise in histone 3, which coincides with mitosis, was only seen after 70% hepatectomy, indicating that after 90% hepatectomy the response to growth-stimulating factors is weak or delayed, supported by a delayed rise in cyclin d and low levels of growth hormone receptor mRNA. CONCLUSIONS: It is concluded that attempts by gene regulation to adapt liver functions to a reduction of the liver mass depend on the amount of liver tissue lost. When the loss is nearly fatal, compensation for normal metabolic functions may be abandoned for efforts to regenerate, which, however, may be delayed or after all be too weak.

Actins↗
PubMed1995

Nitric-oxide-mediated activation of iron-regulatory protein controls hepatic iron metabolism during acute inflammation.

The molecular regulation of intracellular iron metabolism has been studied in the livers of rats undergoing an acute inflammatory reaction following turpentine injection. Treatment induced an increase in the steady-state level of the transferrin receptor (TfR) mRNA, peaking 18 h after treatment and returning to control levels 24 h after treatment, with no change in TfR gene transcription. RNA band-shift assays documented an activation of the cytoplasmic RNA-binding protein called the iron-regulatory protein (IRP), in parallel with a rise in the amount of TfR transcripts. A 2-3-fold increase in the amount of H and L ferritin subunit mRNAs was found 12-18 h after turpentine treatment. Surprisingly, higher accumulation of ferritin mRNAs did not result in appreciable differences in the liver ferritin content. This might be due to the concomitant rise of IRP activity, which is known to prevent ferritin mRNA translation. The absence of significant changes in the total iron and ferritin contents prompted us to investigate the role of nitric oxide (NO), an inflammatory mediator which is also known to modulate the activity of IRP. Northern-blot analysis showed a marked enhancement in the expression of the inducible form of nitric oxide synthase mRNA in turpentine-treated rats. Furthermore, the activation of IRP and the increase of the TfR mRNA content that occur in turpentine-treated rats were abolished by treatment with N5-nitro-L-arginine, a specific nitric oxide synthase inhibitor. The present data suggest that NO-mediated activation of IRP regulates alterations of hepatic iron homeostasis that occur in acute inflammation.

Acute Disease↗
PubMed1995

Duodenal ferritin synthesis in genetic hemochromatosis.

BACKGROUND/AIMS: The molecular defect of genetic hemochromatosis (GH) is unknown. It is believed that low expression of duodenal ferritin in GH is caused by tissue or cell specific defect of ferritin synthesis. Our study was designed to ascertain whether the control of duodenal ferritin synthesis in GH was defective. METHODS: Expression at the single cell level of H and L ferritin messenger RNAs and protein and activity of the iron regulatory factor, which controls the translation of ferritin messenger RNA, were assessed in 43 duodenal biopsy specimens from individuals with GH, secondary hemochromatosis (SH), anemia, or normal iron balance. RESULTS: Signal for ferritin H and L subunit messenger RNAs was detected in both absorptive and nonabsorptive cells by in situ hybridization, but in 10 of 14 patients with untreated GH, the signal was lower than in patients with SH or normal subjects. However, immunostaining for ferritin protein documented a diffuse/cytoplasmic pattern, whereas a supranuclear/granular staining was found in normal subjects or patients with SH. The spontaneous activity of duodenal iron regulatory factor was consistently higher in patients with GH than in normal subjects or subjects with anemia or SH. CONCLUSIONS: In patients with GH, ferritin gene transcription is preserved in both absorptive and nonabsorptive intestinal cells. Low accumulation of ferritin is not caused by a defective control of ferritin synthesis but by low expression of ferritin messenger RNA and sustained activity of iron regulatory factor.

Adolescent↗
PubMed1995

Antioxidant activity of silybin in vivo during long-term iron overload in rats.

BACKGROUND & AIMS: Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. METHODS: Rats were fed a 2.5% carbonyl-iron diet and 100 mg.kg body wt-1.day-1 silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. RESULTS: Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. CONCLUSIONS: Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.

Adenosine Triphosphate↗
PubMed1995

Gene expression of urea cycle enzymes following two-thirds partial hepatectomy in the rat.

The effect of reduction of functional liver mass on the expression of enzyme systems for hepatic urea synthesis was assessed in rats following two-thirds partial hepatectomy. Results were related to normal, fed rats and to sham-operated rats, with identical timing for surgery and feeding. Among the five urea cycle enzymes the mRNA steady-state level was higher in hepatectomized than in sham-operated rats for carbamoyl phosphate synthetase and arginino-succinate lyase. The level for albumin mRNA remained close to that of the controls. Relative transcription rates were found to be increased for carbamoyl phosphate synthetase, arginino-succinate synthase and arginase. For albumin the transcription rate was drastically reduced initially, but recovered gradually during the experimental period. The data indicate that the expression of urea cycle enzymes, in particular that of carbamoyl phosphate synthetase which is the rate-limiting step, is up-regulated by partial hepatectomy. This helps to maintain urea synthesis rate at a normal or near normal level during the period of reduced liver mass, confirming metabolic studies. In contrast, the transcription for albumin was reduced. The immediate increase in urea cycle enzyme expression during the period of acute hepatocyte loss is consistent with the view that it is vitally important that urea synthesis, in contrast to e.g. albumin synthesis, remains intact when the metabolic capacity of the liver is reduced.

Albumins↗
PubMed1995

Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.

The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32-B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

Animals↗
PubMed1995

Molecular and cellular aspects of iron-induced hepatic cirrhosis in rodents.

Hepatic fibrosis and cirrhosis are common findings in humans with hemochromatosis. In this study we investigated the molecular pathways of iron-induced hepatic fibrosis and evaluated the anti-fibrogenic effect of vitamin E. Male gerbils were treated with iron-dextran and fed a standard diet or a alpha-tocopherol enriched diet (250 mg/Kg diet). In gerbils on the standard diet at 6 wk after dosing with iron, in situ hybridization analysis documented a dramatic increase of signal for collagen mRNA around iron foci onto liver fat storing cells (FSC), as identified by immunocytochemistry with desmin antibody. After 4 mo, micronodular cirrhosis developed in these animals, with nonparenchymal cells surrounding hepatocyte nodules and expressing high level of TGF beta mRNA. In this group, in vivo labeling with [3H]-thymidine showed a marked proliferation of nonparenchymal cells, including FSC. In iron-dosed gerbils on the vitamin E-enriched diet for 4 mo, in spite of a severe liver iron burden, a normal lobular architecture was found, with a dramatic decrease of collagen mRNA accumulation and collagen deposition. At the molecular level, a total suppression of nonparenchymal cell proliferation was appreciable, although expression of collagen and TGF beta mRNAs was still present into microscopic iron-filled nonparenchymal cell aggregates scattered throughout the hepatic lobule. In conclusion, our study shows that anti-oxidant treatment during experimental hepatic fibrosis arrests fibrogenesis and completely prevents iron induced hepatic cirrhosis mainly through inhibition of nonparenchymal cell proliferation induced by iron.

Animals↗
PubMed1994

Transferrin receptor gene expression during rat liver regeneration. Evidence for post-transcriptional regulation by iron regulatory factorB, a second iron-responsive element-binding protein.

Transferrin receptor (TfR) expression is regulated by iron at the level of mRNA stability through a factor (IRF/IRE-BP) which binds to specific iron-responsive elements (IRE). On the other hand, growth-dependent regulation of TfR expression is generally believed to be transcriptionally controlled. We analyzed the molecular mechanisms that control TfR gene expression at the onset of cell proliferation in vivo during liver regeneration after partial hepatectomy. The amount of TfR mRNA increased considerably after partial hepatectomy while run-on assays did not show significant changes in TfR gene transcription. RNA band-shift assays documented a significant activation of IRF/IRE-BP specific for the faster migrating IRE-protein complex (IRFB). These changes occurred in the absence of modifications of total liver iron concentration but together with a significant decrease of ferritin content. Moreover, when extreme variations of liver iron content were achieved by either chronic iron overload or severe iron deficiency, liver regeneration was unable to influence IRE-binding activity. We conclude that IRF/IRE-BP-mediated post-transcriptional control can fully account for TfR mRNA induction during liver cell proliferation in vivo. IRF/IRE-BP activation in the absence of changes in total tissue iron content might depend either on a drop of iron levels into the regulatory pool or on a relatively iron-independent mechanism specific for the faster migrating complex.

Animals↗
PubMed1994

Homeostatic regulation of hepatocyte nuclear transcription factor 1 expression in cultured hepatoma cells.

Serum colloid osmotic pressure is believed to control the hepatic output of plasma proteins, including albumin. The present study was aimed at identifying the molecular basis for feedback control of albumin gene expression in highly differentiated hepatoma cells. The steady-state level of albumin mRNA and the activity of a 282-bp albumin promoter-chloramphenicol acetyltransferase reporter gene in cells incubated in the presence of increasing amounts of serum albumin or dextran were significantly and selectively decreased. When nuclear extracts from cells exposed to 5% (wt/vol) serum albumin were tested in a gel-retardation assay with six oligonucleotide probes containing DNA elements of the albumin promoter, only the element B-retarded band, which contains the nucleotide recognition sequence for hepatocyte nuclear transcription factor 1 alpha (HNF-1 alpha), was consistently decreased as compared to nuclear extract from cells not exposed to serum albumin. Moreover, the activity of a reporter gene with a basal TATA-promoter driven by multiple HNF-1 alpha recognition elements was selectively inhibited in cells incubated in the presence of 5% serum albumin. A reduction of HNF-1 alpha mRNA appears to be responsible for this response to a change in the level of macromolecules in the incubation medium. These results indicate that activity of a dominant liver transcription factor, HNF-1 alpha, controlling the transcription of several liver-specific genes, is modulated by a fluctuation in the level of oncotically active macromolecules.

Animals↗
PubMed1994

Enhanced hepatic collagen type I mRNA expression into fat-storing cells in a rodent model of hemochromatosis.

In recent years, identifying the hepatic cell type responsible for collagen synthesis in experimental models of postnecrotic or inflammatory fibrosis has been the subject of active investigation. In primary iron overload states, however, hepatic fibrosis and cirrhosis occur without accompanying necroinflammatory phenomena. In this study, we combined morphological, immunological, cell isolation and purification and molecular biological techniques to identify the hepatic cell responsible for enhanced collagen type I gene expression during chronic enteral iron overload in the rat. Ultrastructural analysis of liver tissue sections from iron-loaded rats specifically revealed an altered appearance of fat-storing cells, which showed few if any fat droplets left and increased rough endoplasmic reticulum. In situ hybridization analysis with specific complementary RNA probes identified enhanced signal for collagen type I into nonparenchymal cells in zones 1 and 2, without signal over the background onto iron-laden hepatocytes. Immunocytochemistry with desmin antibodies combined with in situ hybridization on the same tissue sections identified the cells expressing high level of collagen type I transcripts as fat-storing cells. Northern-blot analysis on RNA extracted from various purified cell isolates, confirmed the presence of collagen type I mRNA signal only into the fat-storing cells isolate. Our study shows that in an experimental model of metabolic fibrosis in which the hepatotoxin selectively accumulates into parenchymal cells, fat-storing cells are the main source of enhanced collagen type I gene expression.

Animals↗