Nuova ricerca

Giuliana MONTOSI

Personale tecnico amministrativo
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

Home |


Pubblicazioni

2023 - CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development [Articolo su rivista]
Vecchi, C.; Montosi, G.; Garuti, C.; Canali, S.; Sabelli, M.; Bergamini, E.; Ricci, A.; Buzzetti, E.; Corradini, E.; Pietrangelo, A.
abstract

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge.Key messagesCREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood.CHEB-H is involved in hepcidin gene response to oral iron in vivo.CREB-H loss hampers hepcidin promoter response to BMP6.CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

2020 - WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis [Articolo su rivista]
Smargiassi, A.; Bertacchini, J.; Checchi, M.; Poti, F.; Tenedini, E.; Montosi, G.; Magaro, M. S.; Amore, E.; Cavani, F.; Ferretti, M.; Grisendi, G.; Maurel, D. B.; Palumbo, C.
abstract

The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

2018 - Wisp2 overexpression induced by short Teriparatide treatment affects IDG-SW3 osteogenic differentiation. [Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Magaro', MARIA SARA; Poti', Francesco; Tenedini, Elena; Montosi, Giuliana; Magaro', MARIA SARA; Vinet, Jonathan; Maurel, Delphine; Palumbo, Carla
abstract

The study supports the importance of osteocytes in controlling the action of the other bone cells and suggests that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

2017 - Human macrophage ferroportin biology and the basis for the ferroportin disease [Articolo su rivista]
Sabelli, Manuela; Montosi, Giuliana; Garuti, Cinzia; Caleffi, Angela; Oliveto, Stefania; Biffo, Stefano; Pietrangelo, Antonello
abstract

Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. Conclusion: FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).

2017 - Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization [Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Tenedini, Elena; Montosi, Giuliana; Vinet, Jonathan; Ferretti, Marzia; Palumbo, Carla
abstract

PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

2016 - The SMAD pathway is required for hepcidin response during endoplasmic reticulum stress [Articolo su rivista]
Canali, Susanna; Vecchi, Chiara; Garuti, Cinzia; Montosi, Giuliana; Babitt, Jodie L; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is regulated by a number of stimulatory and inhibitory signals. The cAMP responsive element binding protein 3-like 3, CREB3L3, mediates hepcidin response to endoplasmic reticulum (ER) stress. In this study we asked whether hepcidin response to ER stress also requires the SMAD1/5/8 pathway that has a major role in hepcidin regulation in response to iron and other stimuli. We analyzed hepcidin mRNA expression and promoter activity in response to ER stressors in HepG2 cells in the presence of the BMP type I receptor inhibitor LDN-193189, mutated hepcidin promoter or siRNA against different SMAD proteins. We then used a similar approach in vivo in wild-type, Smad1/5 or Creb3l3 -/- animals undergoing ER stress. In vitro, LDN-193189 prevented hepcidin mRNA induction by different ER stressors. Seemingly, mutation of a BMP-responsive element in the hepcidin promoter prevented ER stress-mediated upregulation. Moreover, in vitro silencing of SMAD proteins by siRNA, in particular SMAD5, blunted hepcidin response to ER stress. On the contrary, hepcidin induction by ER stress was maintained when using antibodies against canonical BMP receptor ligands. In vivo, hepcidin was induced by ER stress and prevented by LDN-193189. In addition, in Smad1/5 knock-out mice, ER stress was unable to induce hepcidin expression. Finally, in Creb3l3 knock-out mice, in response to ER stress, SMAD1/5 were correctly phosphorylated and hepcidin induction was still appreciable, although to a lesser extent as compared to control mice. In conclusion, our study indicates that hepcidin induction by ER stress involves the central regulatory SMAD1/5 pathway.

2014 - GLUCONEOGENIC SIGNALS DIRECTLY CONTROL IRON HOMEOSTASIS THROUGH HEPCIDIN [Abstract in Atti di Convegno]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Qian, J; Liu, C; Canali, S; Pietrangelo, Antonello
abstract

-

2014 - Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Canali, Susanna; Pietrangelo, Antonello
abstract

Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance).|We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and wild-type and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses.|Starvation led to increased transcription of encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a messenger RNA and Creb3l3 messenger RNA, which encode a transcriptional co-activator involved in energy metabolism and a liverspecific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation.|We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.

2014 - SEX HORMONES DIFFERENTLY REGULATE HEPATIC HEPCIDIN EXPRESSION AND SYSTEMIC IRON HOMEOSTASIS IN VIVO [Abstract in Rivista]
Garuti, Cinzia; Montosi, Giuliana; Barelli, S; Pietrangelo, Antonello; Corradini, Elena
abstract

-

2013 - SEX HORMONES DIFFERENTLY REGULATE HEPCIDIN EXPRESSION AND IRON HOMEOSTASIS IN VIVO. [Abstract in Rivista]
Garuti, Cinzia; Montosi, Giuliana; S., Barelli; Pietrangelo, Antonello; Corradini, Elena
abstract

-

2011 - THE MOLECULAR BASIS FOR THE HEPATIC REGULATION OF HEPCIDIN, THE IRON HORMONE, BY BONE MORPHOGENETIC PROTEINS. [Abstract in Rivista]
Corradini, Elena; Meynard, D; Montosi, Giuliana; Garuti, Cinzia; Wu, Q; Ventura, Paolo; Babitt, Jl; Lin, Hy; Pietrangelo, Antonello
abstract

-

2010 - BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice. [Articolo su rivista]
Corradini, Elena; Schmidt, Pj; Meynard, D; Garuti, Cinzia; Montosi, Giuliana; Chen, S; Vukicevic, S; Pietrangelo, Antonello; Lin, Hy; Babitt, J. L.
abstract

BACKGROUND AND AIMS: Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)-SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently demonstrated to be impaired in Hfe knockout (Hfe(-/-)) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe(-/-) mice.METHODS: The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice (Hfe Tg) compared with wildtype (WT) mice. Hfe(-/-) and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters.RESULTS: Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, while anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe(-/-) mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites.CONCLUSIONS: HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.

2010 - Hepcidin expression does not rescue the iron-poor phenotype of Kupffer cells in Hfe-null mice after liver transplantation. [Articolo su rivista]
Garuti, Cinzia; Tian, Y; Montosi, Giuliana; Sabelli, Manuela; Corradini, Elena; Graf, R; Ventura, Paolo; Vegetti, Alberto; Clavien, Pa; Pietrangelo, Antonello
abstract

BACKGROUND & AIMS: Hemochromatosis is a common hereditary disease caused by mutations in HFE and characterized by increased absorption of iron in the intestine. However, the intestine does not appear to be the site of mutant HFE activity in the disease; we investigated the role of the liver-the source of the iron regulatory hormone hepcidin-in pathogenesis in mice. METHODS: We exchanged livers between Hfe wild-type (+/+) and Hfe null (-/-) mice by orthotopic liver transplantation (OLT) and assessed histopathology, serum and tissue iron parameters, and hepatic hepcidin messenger RNA expression. RESULTS: At 6-8 months after OLT, Hfe(-/-) mice that received Hfe(-/-) livers maintained the hemochromatosis phenotype: iron accumulation in hepatocytes but not Kupffer cells (KC), increased transferrin levels, and low levels of iron in the spleen. Hfe(+/+) mice that received Hfe(-/-) livers had increased levels of iron in serum and liver and low levels of iron in spleen. However, they did not develop the iron-poor KCs that characterize hemochromatosis: KCs appeared iron rich, although hepatic hepcidin expression was low. Transplantation of Hfe(+/+) livers into Hfe(-/-) mice prevented hepatic iron accumulation but did not return spleen and plasma levels of iron to normal; KCs still appeared to be iron poor, despite normal hepcidin expression. CONCLUSIONS: In Hfe(-/-) mice, transplantation of livers from Hfe(+/+) mice reversed the iron-loading phenotype associated with hemochromatosis (regardless of Hfe expression in intestine). However, KCs still had low levels of iron that were not affected by hepatic hepcidin expression. These findings indicate an independent, iron-modifying effect of HFE in KCs.

2010 - Huh-7: a human hemochromatotic cell line. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; Pietrangelo, Antonello
abstract

Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine-to-tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta(2)-microglobulin (beta(2)M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh-7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh-7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HIE was detected by western blot in HepG2 but not in Huh-7 cell membrane fractions. In WRL-68 cells expressing wild-type HIE, the HIE protein was largely found at the plasma membrane where it colocalizes with beta(2)M. On the contrary, the HFE-Y231del mutant, similarly to an exogenously expressed HFE-C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane-expressed beta(2)M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh-7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild-type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh-7 cells stably expressing exogenous wild-type HFE at the plasma membrane. Conclusion: Huh-7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HIE HC. (HEPATOLOGY 2010;51:654-659.)

2009 - Bone Morphogenetic Protein Signaling Is Impaired in an Hfe Knockout Mouse Model of Hemochromatosis. [Articolo su rivista]
Corradini, Elena; Garuti, Cinzia; Montosi, Giuliana; Ventura, Paolo; Andriopoulos, B; Lin, Hy; Pietrangelo, Antonello; Babitt, Jl
abstract

Mutations in HFE are the most common cause of the iron-overload disorder hereditary hemochromatosis. Levels of the main iron regulatory hormone, hepcidin, are inappropriately low in hereditary hemochromatosis mouse models and patients with HFE mutations, indicating that HFE regulates hepcidin. The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is an important endogenous regulator of hepcidin expression. We investigated whether HFE is involved in BMP6-SMAD regulation of hepcidin expression. METHODS: The BMP6-SMAD pathway was examined in Hfe knockout (KO) mice and in wild-type (WT) mice as controls. Mice were placed on diets of varying iron content. Hepcidin induction by BMP6 was examined in primary hepatocytes from Hfe KO mice; data were compared with those of WT mice. RESULTS: Liver levels of Bmp6 messenger RNA (mRNA) were higher in Hfe KO mice; these were appropriate for the increased hepatic levels of iron in these mice, compared with WT mice. However, levels of hepatic phosphorylated Smad 1/5/8 protein (an intracellular mediator of Bmp6 signaling) and Id1 mRNA (a target gene of Bmp6) were inappropriately low for the body iron burden and Bmp6 mRNA levels in Hfe KO, compared with WT mice. BMP6 induction of hepcidin expression was reduced in Hfe KO hepatocytes compared with WT hepatocytes. CONCLUSIONS: HFE is not involved in regulation of BMP6 by iron, but does regulate the downstream signals of BMP6 that are triggered by iron.

2009 - ER stress controls iron metabolism through induction of hepcidin. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; K., Zhang; Lamberti, Igor; S. A., Duncan; R. J., Kaufman; Pietrangelo, Antonello
abstract

Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin overproduction causes anemia of inflammation, whereas its deficiency leads to hemochromatosis. Inflammation and iron are known extracellular stimuli for hepcidin expression. We found that endoplasmic reticulum (ER) stress also induces hepcidin expression and causes hypoferremia and spleen iron sequestration in mice. CREBH (cyclic AMP response element-binding protein H), an ER stress-activated transcription factor, binds to and transactivates the hepcidin promoter. Hepcidin induction in response to exogenously administered toxins or accumulation of unfolded protein in the ER is defective in CREBH knockout mice, indicating a role for CREBH in ER stress-regulated hepcidin expression. The regulation of hepcidin by ER stress links the intracellular response involved in protein quality control to innate immunity and iron homeostasis.

2007 - Effect of anti-osteoarthritic drugs on interleukin 1-dependent activation of NF-kappa B in human chondrocytes [Abstract in Atti di Convegno]
S., Barelli; Garuti, Cinzia; Montosi, Giuliana; Pietrangelo, Antonello
abstract

No abstract available

2005 - Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload [Articolo su rivista]
Montosi, Giuliana; Corradini, Elena; Garuti, Cinzia; S., Barelli; S., Recalcati; G., Cairo; L., Valli; Pignatti, Elisa; Vecchi, Chiara; F., Ferrara; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl3) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.

2005 - Lack of enterocyte iron accumulation in the ferroportin disease [Articolo su rivista]
Corradini, Elena; Montosi, Giuliana; F., Ferrara; A., Caleffi; Pignatti, Elisa; S., Barelli; Garuti, Cinzia; Pietrangelo, Antonello
abstract

Ferroportin-associated iron overload (also known as the ferroportin disease) is a common cause of hereditary hyperferritinemia. It was originally proposed that loss-of-protein function mutations account for iron overload in the FD. This hypothesis is consistent with the 14 phenotype reported in most patients with FD of early iron accumulation in tissues, particularly in macrophages, in spite of relatively normal-low circulatory iron. It was still unclear, however, how FPN mutations would affect iron retention in enterocytes. We studied histologically the intestine of six patients with different FPN mutations as compared to other subjects with various iron disorders. We found that regardless of the underlying FPN mutation, no iron accumulation was found in absorbing enterocytes while, intestinal villi showed marked signs of iron accumulation in the cells of lamina propria. Not surprisingly, in the liver, iron excess was found mainly in Kupffer cells. These results indicate that FPN haploinsufficiency is not limiting for iron export from enterocytes.

2004 - Erratum: Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene (Blood Cells, Molecules, and Diseases (2003) 31 (299-304) DOI: 10.1016/S1079-9796(03)00164-5) [Articolo su rivista]
Gordeuk, V. R.; Caleffi, A.; Corradini, E.; Ferrara, F.; Jones, R. A.; Castro, O.; Onyekwere, O.; Kittles, R.; Pignatti, E.; Montosi, G.; Garuti, C.; Gangaidzo, I. T.; Gomo, Z. A. R.; Moyo, V. M.; Rouault, T. A.; Macphail, P.; Pietrangelo, A.
abstract

2003 - Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene [Articolo su rivista]
Gordeuk, Vr; Caleffi, Angela; Corradini, Elena; Ferrara, Francesca; Jones, Ra; Castro, O; Onyekwere, O; Kittles, R; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Gangaidzo, It; Gomo, Zar; Moyo, Vm; Rouault, Ta; Macphail, P; Pietrangelo, Antonello
abstract

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N = 15) and the United States (N = 4) presenting with primary iron overload. We found a new c. 744 C→T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 μg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 μg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.

2003 - The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression [Articolo su rivista]
A., Lymboussaki; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Dj, Haile; Pietrangelo, Antonello
abstract

Background/Aims: MTP1/Ferroportin1/IREG1, the product of the SLC40A1 gene, is a main iron export protein in mammals. However, the way this gene is regulated by iron is still unclear. The aim of this study was to investigate the functional role of genomic SLC40A1 elements in response to iron. Methods: Vectors containing either similar to 2.6 kb 5' flanking region or deletion constructs, including one devoid of an iron responsive element (SLC40A1-DeltaIRE-Luc), were analyzed by luciferase reporter gene in transfected HepG2, CaCO2 and U937 cells. Expression of iron genes and activity of the iron regulatory protein were also studied. Results: Iron increased and desferrioxamine decreased luciferase activity in all the cell types using both the full-length construct and the promoter deletion constructs, in the absence of changes in SLC40A1 or luciferase mRNA levels. To test the role of the SLC40A1 5' untranslated region, we first demonstrated that wild type and not SLC40A1-DeltaIRE-Luc could bind iron regulatory protein. Then, in cells transfected with SLC40A1-DIRE-Luc, we found that, in spite of iron regulatory protein activation, the response to iron manipulation was lost. Conclusions: We demonstrate that the iron responsive element in the SLC40A1 gene is functional and that it controls gene expression through the cytoplasmic iron regulatory protein system. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

2002 - Iron-induced oxidant stress in nonparenchymal liver cells: Mitochondrial derangement and fibrosis in acutely iron-dosed gerbils and its prevention by silybin [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Contri, Miranda; F., Giovannini; D., Ceccarelli; A., Masini
abstract

Hepatic fibrosis due to iron overload is mediated by oxidant stress. The basic mechanisms underlying this process in vivo are still little understood. Acutely iron-dosed gerbils were assayed for lobular accumulation of hepatic lipid peroxidation by-products, oxidant-stress gene response, mitochondrial energy-dependent functions, and fibrogenesis. Iron overload in nonparenchymal cells caused an activation of hepatic stellate cells and fibrogenesis. Oxidant-stress gene response and accumulation of malondialdehyde-protein adducts were restricted to iron-filled nonparenchymal cells, sparing nearby hepatocytes. Concomitantly, a significant rise in the mitochondrial desferrioxamine-chelatable iron pool associated with the impairment of mitochondrial oxidative metabolism and the hepatic ATP decrease, was detected. Ultrastructural mitochondrial alterations were observed only in nonparenchymal cells. All biochemical and functional derangements were hindered by in vivo silybin administration which blocked completely fibrogenesis. Iron-induced oxidant stress in nonparenchymal cells appeared to bring about irreversible mitochondrial derangement associated with the onset of hepatic fibrosis.

2001 - Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene [Articolo su rivista]
Montosi, Giuliana; Donovan, A.; Totaro, Antonella; Garuti, Cinzia; Pignatti, Elisa; Cassanelli, S.; Trenor, C. C.; Gasparini, P.; Andrews, N. C.; Pietrangelo, Antonello
abstract

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

2001 - Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin 1 in iron deficiency and iron overload [Articolo su rivista]
Zoller, H; Koch, R; Theurl, I; Obrist, P; Pietrangelo, Antonello; Montosi, Giuliana; Haile, D; Vogel, W; Weiss, G.
abstract

Background & AIMS: Imbalances of iron homeostasis are accompanied by alterations of intestinal iron absorption. The identification of divalent-metal transporter 1 (DMT1) and ferroportin 1 (FP1) has improved our understanding of transmembrane iron trafficking. To gain insight into the regulatory properties of these transporters in the duodenum, we studied their expression in patients with hereditary hemochromatosis (HFE-associated and non-HFE-associated), secondary iron overload, and iron deficiency. Methods: DMT1, FP1 messenger RNA (mRNA), and protein expression were analyzed in duodenal biopsy specimens from patients by means of TaqMan real-time polymerase chain reaction, Western blotting technique, and immunohistochemistry. Results: DMT1 and FP1 mRNA levels are positively correlated with each other in all patient groups (P < 0.001), Moreover, DMT1 and FP1 mRNA levels were significantly increased in patients with iron deficiency, HFE and non-HFE hemochromatosis, whereas they were unchanged in patients with secondary iron overload. Alterations in DMT1 and FP1 mRNA levels were paralleled by comparable changes in the duodenal expression of these proteins. In patients with normal iron status or iron deficiency, significant negative correlations between DMT1, FP1 tnRNA, and serum iron parameters were found, which were absent in subjects with primary hemochromatosis, Conclusions: DMT1 and FP1 are centrally involved in iron uptake/transfer in the duodenum and in the adaptive changes of iron homeostasis to iron deficiency and overload.

2001 - Frequency and biochemical expression of C282Y/H63D hemochromatosis (HFE) gene mutations in the healthy adult population in Italy [Articolo su rivista]
Cassanelli, S; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Mariano, M; Campioli, D; Carbonieri, A; Baldini, Enrica; Pietrangelo, Antonello
abstract

Background/Aims: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. Methods: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. Results: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. Conclusions: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

2000 - Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin [Articolo su rivista]
A., Masini; D., Ceccarelli; F., Giovannini; Montosi, Giuliana; Garuti, Cinzia; Pietrangelo, Antonello
abstract

Hepatic iron toxicity because of iron overload seems to be mediated by lipid peroxidation of biological membranes and the associated organelle dysfunctions. However, the basic mechanisms underlying this process in vivo are still little understood. Gerbils were dosed with weekly injections of iron-dextran alone or in combination with sylibin, a well-known antioxidant, by gavage for 8 weeks. A strict correlation was found between lipid peroxidation and the level of desferrioxamine chelatable iron pool. A consequent derangement in the mitochondrial energy-transducin capability, resulting from a reduction in the respiratory chain enzyme activities, occurred. These irreversible oxidative anomalies brought about a dramatic drop in tissue ATP level, The mitochondrial oxidative derangement was associated with the development of fibrosis in the hepatic tissue. Silybin administration significantly reduced both functional anomalies and the fibrotic process by chelating desferrioxamine chelatable iron.

2000 - Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis [Articolo su rivista]
Montosi, Giuliana; P., Paglia; Garuti, Cinzia; Ca, Guzman; Jm, Bastin; Mp, Colombo; Pietrangelo, Antonello
abstract

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we stud led monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of Fe-55 delivered by Fe-55-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface, The accumulation of Fe-55 delivered by 55Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the Fe-55-ferritin pool within the HFE-transfected cells. These results suggest that the Iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

1999 - Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; A., Totaro; Garuti, Cinzia; D., Conte; Cassanelli, Stefano; M., Fraquelli; C., Sardini; F., Vasta; P., Gasparini
abstract

Background and Methods Hereditary hemochromatosis in adults is usually characterized by mutations in the hemochromatosis (HFE) gene on the short arm of chromosome 6. Most patients have a substitution of tyrosine for cysteine at position 282 (C282Y). We studied a large family from Italy that includes persons who have a hereditary iron-overload condition indistinguishable from hemochromatosis but without apparent pathogenic mutations in the HFE gene. We performed biochemical, histologic, and genetic studies of 53 living members of the family, including microsatellite analysis of chromosome 6 and direct sequencing of the HFE gene, Results Of the 53 family members, 15 had abnormal serum ferritin levels, values for transferrin saturation that were higher than 50 percent, or both. Thirteen of the 15 had elevated body iron levels, diagnosed on the basis of the clinical evaluation and liver biopsy, and underwent iron-removal therapy. The other two, both children, did not undergo liver biopsy or iron-removal therapy. None of the 15 members had the C282Y mutation of the HFE gene; 5 of the 15 (as well as 5 healthy relatives) had another mutation of this gene, a substitution of aspartate for histidine at position 63, but none were homozygous for it. No other mutations were found after sequencing of the entire HFE gene for all family members. Microsatellite analysis showed no linkage of the hemochromatosis phenotype with the short arm of chromosome 6, the site of the HFE gene. Conclusions Hereditary hemochromatosis can occur in adults who do not have pathogenic mutations in the hemochromatosis gene. (N Engl J Med 1999;341:725-32,) (C)1999, Massachusetts Medical Society.

1998 - Diacerhein blocks iron regulatory protein activation in inflamed human monocytes [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; Recalcati, S; Garuti, Cinzia; Cairo, G.
abstract

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.

1998 - Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents [Articolo su rivista]
Montosi, Giuliana; Garuti, Cinzia; S., Martinelli; Pietrangelo, Antonello
abstract

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosis in vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination of in situ hybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that, in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells.

1998 - Heterogeneity of hemochromatosis in Italy [Articolo su rivista]
Piperno, A; Sampietro, M; Pietrangelo, Antonello; Arosio, C; Lupica, L; Montosi, Giuliana; Vergani, A; Fraquelli, M; Girelli, D; Pasquero, P; Roetto, A; Gasparini, P; Fargion, Silvia; Conte, Daniele; Camaschella, C.
abstract

Background & Aims: Patients with hemochromatosis show variable phenotype expression. We evaluated the frequency of hemochromatosis gene (HFE) mutations and the contribution of HFE genotype, ancestral haplotype, ethnic background, and additional factors (alcohol intake, hepatitis viruses, and beta-thalassemia trait) to the severity of iron overload in a large series of Italian patients with a hemochromatosis phenotype. Methods: HFE genotype was studied in 188 patients. Phenotype evaluation was available in 153 men and 20 women and was based mainly on iron removed. HFE genotype was determined by a polymerase chain reaction restriction assay and ancestral haplotype through D6S265 and D6S105 microsatellite analysis. Results: The frequency of C282Y homozygotes was 64%, with a decreasing gradient from north to south. C282Y homozygotes showed more severe iron overload than the other HFE genotypes. In the same group, ancestral haplotype was associated with a more severe phenotype. Additional factors may favor the development of a relatively mild hemochromatosis phenotype in patients nonhomozygous for the C282Y mutation. Conclusions: Hemochromatosis in Italy is a nonhomogenous disorder in which genetic and acquired factors are involved. In patients with a single or no HFE mutation, further studies will enable a differentiation between true genetic disorders and interactions between genetic and acquired factors.

1998 - No HFE, no HLA, no 6p-linked adult hemochromatosis: A new genetic iron overload condition [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Conte, D; Fraquelli, M; Gasparini, P.
abstract

-

1998 - Oxidant stress: cell signalling and gene response in the liver: the iron-oxygen connection. [Relazione in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; S., Martinelli
abstract

-

1998 - Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis [Articolo su rivista]
Montosi, Giuliana; Garuti, Cinzia; Iannone, Anna; Pietrangelo, Antonello
abstract

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.

1998 - Ursodeoxycholic acid complexation with 2-hydroxypropyl-beta-cyclodextrin increases ursodeoxycholic acid biliary excretion after single oral administration in rats [Abstract in Rivista]
Ventura, Paolo; Panini, Rossana; Montosi, Giuliana; Garuti, Cinzia; Vandelli, Maria Angela; G., Brunetti; Pietrangelo, Antonello; Salvioli, Gianfranco
abstract

Complexation of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta-cyclodextrin (HPbCD) (1:1 molar ratio) improves the water solubility and dissolution rate of UDCA and hence its bioavailability. We compared UDCA bile recovery (percentage of administered UDCA dose excreted in bile) after single oral (gavage) administration of UDCA in three different pharmaceutical forms in a bile fistula rat model. Male Sprague-Dawley rats were randomly assigned to 11 groups (6 rats per groups); the bile duct was cannulated after gavage to collect 4-h bile samples. Groups 1, 2 and 3 (fed 5, 10 and 50 mg/kg body weight UDCA/HPbCD complex, respectively) showed (mean values ± SD) 39.2 ± 5.9%, 25.7 ± 4.1% and 9.4 ± 3.1% UDCA bile recovery, respectively; in groups 4, 5 and 6 (fed UDCA suspension formulation containing 5, 10 and 50 mg/kg body weight, respectively) UDCA bile recovery was 33.2 ± 3.6%, 16.9 ± 4.7% and 6.3 ± 0.8%, respectively; groups 7, 8 and 9 (fed UDCA capsule formulation containing 5, 10 and 50 mg/kg body weight, respectively) showed 30.6 ± 9.0%, 21.7 ± 6.4% and 4.7 ± 1.8% UDCA recovery. Groups 10 and 11 (controls) were fed with HPbCD and saline, respectively. Results indicate a significantly (p<0.05) higher bile UDCA recovery in rats fed UDCA-HPbCD complex than in those fed UDCA suspension or UDCA capsule formulations at the same dose. In this model, oral UDCA/HPbCD complex increased UDCA biliary excretion making for greater UDCA enrichment of the bile acid pool than identical doses of common pharmaceutical formulations.

1997 - HLA-H mutations in Italian patients with genetic hemochromatosis: Evidence of genetic heterogeneity [Relazione in Atti di Convegno]
Piperno, A; Sampietro, M; Arosio, C; Lupica, L; Vergani, A; Pietrangelo, Antonello; Conte, D; Pasquero, P; Nicoli, C; Montosi, Giuliana
abstract

-

1997 - Inappropriately high iron regulatory protein activity in monocytes of patients with genetic hemochromatosis [Articolo su rivista]
Cairo, G; Recalcati, S; Montosi, Giuliana; Castrusini, E; Conte, D; Pietrangelo, Antonello
abstract

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.

1996 - C/EBP-beta/LAP controls down-regulation of albumin gene transcription during liver regeneration [Articolo su rivista]
Trautwein, C; Rakemann, T; Pietrangelo, Antonello; Plumpe, J; Montosi, Giuliana; Mann, Mp
abstract

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 downregulation 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 G(1) 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 ifs 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 G(1)/S phase checkpoint, down-regulates a major liver function, i.e. albumin synthesis, to prepare the hepatocyte for entry into the cell cycle.

1996 - Defective control or iron metabolism in pre-neoplastic liver nodules during experimental carcinogenesis [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Stal, P; Eriksson, L.
abstract

-

1995 - Antioxidant activity of silybin in vivo during long-term iron overload in rats [Articolo su rivista]
Pietrangelo, Antonello; F., Borella; Casalgrandi, Giovanna; Montosi, Giuliana; D., Ceccarelli; D., Gallesi; F., Giovannini; A., Gasparetto; Masini, Alberto
abstract

Background &amp; 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.

1995 - Duodenal ferritin synthesis in genetic hemochromatosis [Articolo su rivista]
Pietrangelo, Antonello; Casalgrandi, Giovanna; Quaglino, Daniela; R., Gualdi; D., Conte; S., Milani; Montosi, Giuliana; Ventura, Ezio; L., Cesarini; G., Cairo
abstract

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.

1995 - MOLECULAR AND CELLULAR ASPECTS OF IRON-INDUCED HEPATIC CIRRHOSIS IN RODENTS [Articolo su rivista]
Pietrangelo, Antonello; Gualdi, Rossana; Casalgrandi, Giovanna; Montosi, Giuliana; Ventura, Ezio
abstract

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 [H-3]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.

1995 - MOLECULAR AND CELLULAR ASPECTS OF LIVER-DISEASE IN GENETIC HEMOCHROMATOSIS [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Stal, P; Hultcrantz, R.
abstract

-

1994 - Enhanced hepatic collagen type I mRNA expression into fat-storing cells in a rodent model of hemochromatosis. [Articolo su rivista]
Pietrangelo, Antonello; Gualdi, R; Casalgrandi, Giovanna; Geerts, A; DE BLESER, P; Montosi, Giuliana; Ventura, Ezio
abstract

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.

1994 - Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis [Articolo su rivista]
R., Gualdi; Casalgrandi, Giovanna; Montosi, Giuliana; Ventura, Ezio; Pietrangelo, Antonello
abstract

Background/Aims: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity. Methods: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situ hybridization analyses on liver samples were performed. Results: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and ii. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis. Conclusions: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during long-term iron overload in rodents.

1994 - IRON IS A MITOGENIC AND PROFIBROGENIC FACTOR IN EXPERIMENTAL LIVER FIBROSIS DUE TO IRON OVERLOAD [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Gualdi, Rossana; Casalgrandi, Giovanna; Montosi, Giuliana; Ventura, Ezio
abstract

-

1994 - Iron overload in rodents leads to hepatic cirrhosis through enhancement of collagen gene expression into non-parenchymal cells. [Capitolo/Saggio]
Pietrangelo, Antonello; Gualdi, Rossana; G., Casalgrandi; Montosi, Giuliana; Ventura, Ezio
abstract

-

1993 - VITAMIN-E SUPPLEMENTATION PREVENTS HEPATIC CIRRHOSIS DUE TO IRON OVERLOAD IN RODENTS [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Gualdi, Rossana; Casalgrandi, Giovanna; Montosi, Giuliana; Ventura, Ezio
abstract

-