Biomedical subjects

Cinzia Lilli

Publications and source records attributed to Cinzia Lilli.

14 recordsLinked to original sources

PubMed2006

Desmoid and fibroma tumors differently respond to TGFbeta(1) stimulus and ECM macromolecule accumulation.

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.

Blotting, Northern↗

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

Human desmoid fibroblasts: matrix metalloproteinases, their inhibitors and modulation by Toremifene.

BACKGROUND: Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts. METHODS: We investigated collagen accumulation by 3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis. RESULTS: Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts. CONCLUSION: The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation.

Antineoplastic Agents, Hormonal↗

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↗

PubMed2003

Biocompatibility of collagen membranes crosslinked with glutaraldehyde or diphenylphosphoryl azide: an in vitro study.

Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen.

Azides↗

PubMed2003

Silica, hyaluronate, and alveolar macrophage functional differentiation.

BACKGROUND: Silicosis is mediated by macrophages, their soluble mediators, and extracellular matrix molecules. In this study, we investigated the effects of silica and/or hyaluronate (HA) on several alveolar macrophage responses. METHODS: We evaluated glycosaminoglycan (GAG) production by radiolabeled precursors, nitric oxide (NO) release by its oxidation product, phagocytic activity by Candida albicans internalization, and the secretion of two fibrogenic cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta, by specific assays. RESULTS: Silica significantly reduced GAG secretion, particularly HA secretion. Alone, it decreased Candida uptake; associated with HA, it enhanced the reduction. Silica and Candida reduced NO release, which was not significantly affected when silica- or Candida-exposed cells were also treated with HA. TNF-alpha and TGF-beta activities were stimulated by silica but reduced by HA. CONCLUSIONS: The results suggest that silica and HA modify alveolar macrophage functional differentiation. Silica- and HA-induced modifications of the microenvironment could determine whether the response proceeds toward healing and repair or toward lung chronic pathology.

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↗

PubMedDate not supplied

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Analysis of Variance↗