Biomedical subjects

Tiziano Baroni

Publications and source records attributed to Tiziano Baroni.

12 recordsLinked to original sources

PubMed2006

Unique human CD133+ leukemia cell line and its modulation towards a mesenchymal phenotype by FGF2 and TGFbeta1.

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFbeta1. When FGF2 and/or TGFbeta1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFbeta1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases.

AC133 Antigen↗

PubMed2005

P253R fibroblast growth factor receptor-2 mutation induces RUNX2 transcript variants and calvarial osteoblast differentiation.

Unregulated fibroblast growth factor 2 (FGF2) signaling caused by mutations in the fibroblast growth factor receptor (FGFR2) leads to human craniosynostosis such as the Apert syndrome. In an in vitro control model of calvarial osteoblasts from Apert patients carrying the FGFR2 P253R mutation, we studied the changes in cellular phenotype and evaluated the effects of FGF2. Compared with wild-type controls, osteocalcin mRNA was down-regulated in Apert osteoblasts, Runt-related transcription factor-2 (RUNX2) mRNA was differentially spliced, and FGF2 secretion was greater. Total protein synthesis, fibronectin and type I collagen secretion were up-regulated, while protease and glycosidase activities and matrix metalloproteinase-13 (MMP-13) transcription were decreased, suggesting an altered ECM turnover. Adding FGF2 increased protease and glycosidase activities and down-regulated fibronectin and type I collagen secretion in Apert osteoblasts. High affinity FGF2 receptors were up-regulated in Apert osteoblasts and analysis of signal transduction showed elevated levels of Grb2 tyrosine phosphorylation and the Grb2-p85 beta association, which FGF2 stimulation strongly reduced. All together these findings suggest increased constitutive receptor activity in Apert mutant osteoblasts and an autocrine loop involving the FGF2 pathway in modulation of Apert osteoblast behavior.

Acrocephalosyndactylia↗

PubMed2005

Apert and Crouzon syndromes: clinical findings, genes and extracellular matrix.

Apert and Crouzon syndromes are well known craniostenosis. In the last 10 years several studies were performed to provide a better understanding of the etiology and pathogenesis of these diseases. Both have an autosomal dominant mode of transmission, and a mutation in the gene encoding for the fibroblast growth factor receptor 2 (FGFR2) is the cause in most patients. However, the fact that the same mutation can produce a wide range of phenotypic expression makes the mechanism of anomalous development more complex. The extracellular matrix (ECM) is composed of proteins, glycosaminoglycans, and cytokines that are secreted in an autocrine and paracrine manner and are able to modify the ECM. Fibroblast growth factors are complexed with heparan sulfate, a component of the ECM, before binding the FGFR2. Data exist about different expressions of cytokines and ECM macromolecule in craniostenosis-derived fibroblasts and osteoblasts. Changes in ECM composition could explain the altered osteogenic process and account for pathologic variations in cranial development in addition to the FGFR2 mutations.

Acrocephalosyndactylia↗

PubMed2005

Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo results in NOD mice.

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

Age Factors↗

PubMed2003

Mitogenic effects of Brazilian arthropod venom on isolated islet beta cells: in vitro morphologic ultrastructural and functional studies.

BACKGROUND: One of the major pitfalls associated with use of isolated adult islets of Langerhans' cells is their minimal mitotic capacity. Consequently, maintenance of a steady viable islet cell mass is very difficult. To explore how to enhance beta-cell mitogenesis, we have examined the effects of venom fractions extracted from a Brazilian scorpion on morphologic and functional beta-cell patterns. The venom was previously known to induce nesidioblastosis-like effects with chronic hypoglycemia and pancreatitis in animal models. METHODS: Venom fractions purified from Tityus bahiensis were incubated with batches of isolated rat islets, while a morphologic examination, glucose-stimulated insulin release, insulin content, and insulin messenger ribonucleic acid (mRNA) were carried out early during incubation. On fixation and double fluorescence immunolabeling (rhodamine for anti-insulin monoclonal antibodies; fluorescein for anti-5-bromodeoxyuridine), the preparations were imaged by confocal laser microscopy (CLM) for morphometric quantification of the mitoses. Insulin recovery and mRNA were also assessed at 21 days of culture. RESULTS: Under CLM examination, the beta-cell mitotic rate significantly rose from 1 to 12.8% for the venom-exposed islets. At day 7, insulin release and content were significantly lower for the venom-exposed than the control islets. However, at day 21 of culture, insulin release in response to static incubation with glucose and insulin mRNA from the venom-exposed islets was higher than controls (p < .05). CONCLUSIONS: Incubation with the scorpion venom induced a rapid and significant increase in the beta-cell proliferation not associated with a short-term increase in insulin secretion. The latter fully resumed and overcame controls later in culture, possibly after completion of the beta-cell expansion process.

Animals↗

PubMed2003

Cross-talk between interleukin-6 and transforming growth factor-beta3 regulates extracellular matrix production by human fibroblasts from subjects with non-syndromic cleft lip and palate.

BACKGROUND: Transforming growth factor-beta (TGF-beta) interference with interleukin 6 (IL-6) activity and the role of the latter in early human embryonic development prompted us to examine the effects IL-6 on matrix synthesis and the effects of TGF-beta3 on IL-6 expression human cleft lip and palate (CLP) fibroblasts. METHODS: Collagen and glycosaminoglycan (GAG) synthesis were determined by radiolabeled precursors and biglycan expression by Northern blotting before and after adding IL-6. The effects of TGF-beta3 on IL-6 production were assayed by evaluating IL-6 transcript by Northern blotting and IL-6 protein secretion by enzyme-linked immunosorbent assay. RESULTS: The results showed that IL-6 elicited an inhibitory effect on collagen and GAG levels in CLP fibroblasts by lowering hyaluronan and dermatan sulfate secretion. IL-6 up-regulated biglycan expression, but less strongly than TGF-beta3. TGF-beta3 significantly down-regulated IL-6 transcript and secretion in CLP fibroblasts. CONCLUSIONS: These data suggest the increase in matrix components that characterize the CLP fibroblast phenotype might be due to a concerted TGF-beta3-IL-6 action. We hypothesize changes in cross-talk between TGF-beta3 and IL-6 signal transduction pathways are involved in the induction of cleft palate.

Analysis of Variance↗

PubMed2002

Crouzon's syndrome: differential in vitro secretion of bFGF, TGFbeta I isoforms and extracellular matrix macromolecules in patients with FGFR2 gene mutation.

In the Crouzon's syndrome the cranial morphogenic processes are altered due to the early fusion of cranial sutures. We analysed the phenotype of cultured fibroblasts from normal subjects and from Crouzon patients with a specific fibroblast growth factor receptor 2 mutation resulting in a Cys 342 Tyr substitution within the third immunoglobulin domain. Crouzon fibroblasts differed from normal fibroblasts in their extracellular matrix macromolecule accumulation. In Crouzon fibroblasts glycosaminoglycans and fibronectin were decreased and type I collagen increased. As transforming growth factors beta (TGF beta) and basic fibroblasts growth factor (bFGF) together regulate extracellular matrix deposition, we evaluated TGF beta(1), TGF beta(3) and bFGF production by Crouzon and normal fibroblasts. TGF beta(1), TGFb(3) and bFGF levels were lower while TGF beta(1) mRNA transcripts were higher in Crouzon cells. As the increased TGF beta(1) gene expression did not translate into a parallel increase of secreted TGF beta(1), control of TGF beta(1) secretion may be mainly post-transcriptional. Furthermore, adding bFGF increased TGF beta(1) and TGF beta(3) secretion, suggesting the drop may be due to the altered signal transduction of bFGF. These innovative data suggest the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place.

Blotting, Northern↗

PubMed2002

Effects of transforming growth factor-beta1 and tumour necrosis factor-alpha on cultured fibroblasts from skin fibroma as modulated by toremifene.

To determine how toremifene, an anti-oestrogen triphenylethylene derivate, reduces tumour mass, we investigated its modulation of TGF-beta1 and TNF-alpha in fibroma fibroblasts. Normal and fibroma fibroblasts, isolated from patients affected by Gardner's syndrome without or with fibroma manifestation, were cultured in vitro. Secretion of GAG, collagen and TGF-beta1 was increased in fibroma fibroblasts compared to healthy cells. The increase in TGF-beta1 secretion into the medium was associated with a parallel increase in TGF-beta1 gene expression and receptor number. Receptor cross-linking studies using radiolabelled TGF-beta1 revealed more receptors, particularly types I and II, in fibroma fibroblasts than in normal cells. Normal and fibroma fibroblasts did not synthesise TNF-alpha, but they had TNF-alpha membrane receptors, as shown by TNF-alpha assay. TNF-alpha secreted by human monocytes, which may be present in the peritumoral area, increased cell proliferation and GAG accumulation and was, in turn, enhanced by TGF-beta1 treatment. Both growth factors increased angiogenesis, as shown by the CAM assay. Toremifene reduced TGF-beta1 secretion by fibroma fibroblasts and TNF-alpha secretion by monocytes, thus downregulating cell proliferation, ECM macromolecule accumulation and angiogenic progression. We hypothesise that increased TGF-beta1 gene expression and TGF-beta1 secretion in fibroma fibroblasts as well as the subsequent rise in TNF-alpha production by monocytes may facilitate fibroma growth and that toremifene inhibits autocrine and paracrine growth factor production.

Animals↗

PubMed2002

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation.

BACKGROUND: We focused on the phenotype of non-mineralizing MG 63 and mineralizing TE 85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated. MATERIALS AND METHODS: Osteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay. RESULTS: Osteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG 63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE 85 cell proliferation and reduced TE 85 procollagen and osteocalcin production. CONCLUSIONS: The different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG 63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

Alkaline Phosphatase↗

PubMed2002

Expression profiles of craniosynostosis-derived fibroblasts.

BACKGROUND: Craniosynostosis syndromes, a group of connective disorders characterized by abnormalities in vault osteogenesis and premature fusion of bone sutures, are associated with point mutations in FGF receptor family members. The cellular phenotype is characterized by abnormal extracellular matrix turnover. MATERIAL AND METHODS: We used primary cultures of periosteal fibroblasts derived from two different craniosynostosis syndromes, the Apert and Crouzon syndromes. The FGFR2 third immunoglobulin-like domain and its flanking linker regions were analyzed for mutation. DNA microarrays containing 19,200 cDNAs were used to study the gene expression profiles of Apert and Crouzon fibroblasts. The pathologic cells were compared to wild-type human periosteal fibroblasts. RESULTS: The P253R missense mutation and the G338R mutation were observed in Apert and Crouzon fibroblasts, respectively. The genetic profiles, as evaluated by DNA microarrays, yielded different clusters of expressed sequence tag (ESTs) expression within the experiment. Expression profiles from craniosynostosis-derived fibroblasts differ from those of wild-type fibroblasts (288 human ESTs, p< 0.01, pFDR = 0.12). Furthermore, two ESTs clusters discriminate the Crouzon from Apert fibroblasts. The differentially expressed genes cover a broad range of functional activities, including (1) bone differentiation, (2) cell-cycle regulation, (3) apoptotic stimulation, and (4) signaling transduction, cytoskeleton, and vesicular transport. CONCLUSIONS: The transcriptional program of craniosynostosis fibroblasts differs from that of wild-type fibroblasts. Expression profiles of Crouzon and Apert fibroblasts can also be distinguished by two EST expression clusters, thus hinting at a different genetic background.

Acrocephalosyndactylia↗

PubMedDate not supplied

Retinoic acid, GABA-ergic, and TGF-beta signaling systems are involved in human cleft palate fibroblast phenotype.

During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.

Cell Count↗