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Gabriella Fibbi

Publications and source records attributed to Gabriella Fibbi.

13 recordsLinked to original sources
PubMed2006

A model of anti-angiogenesis: differential transcriptosome profiling of microvascular endothelial cells from diffuse systemic sclerosis patients.

The objective of this work was to identify genes involved in impaired angiogenesis by comparing the transcriptosomes of microvascular endothelial cells from normal subjects and patients affected by systemic sclerosis (SSc), as a unique human model disease characterized by insufficient angiogenesis. Total RNAs, prepared from skin endothelial cells of clinically healthy subjects and SSc patients affected by the diffuse form of the disease, were pooled, labeled with fluorochromes, and hybridized to 14,000 70 mer oligonucleotide microarrays. Genes were analyzed based on gene expression levels and categorized into different functional groups based on the description of the Gene Ontology (GO) consortium to identify statistically significant terms. Quantitative PCR was used to validate the array results. After data processing and application of the filtering criteria, the analyzable features numbered 6,724. About 3% of analyzable transcripts (199) were differentially expressed, 141 more abundantly and 58 less abundantly in SSc endothelial cells. Surprisingly, SSc endothelial cells over-express pro-angiogenic transcripts, but also show up-regulation of genes exerting a powerful negative control, and down-regulation of genes critical to cell migration and extracellular matrix-cytoskeleton coupling, all alterations that provide an impediment to correct angiogenesis. We also identified transcripts controlling haemostasis, inflammation, stimulus transduction, transcription, protein synthesis, and genome organization. An up-regulation of transcripts related to protein degradation and ubiquitination was observed in SSc endothelial cells. We have validated data on the main anti-angiogenesis-related genes by RT-PCR, western blotting, in vitro angiogenesis and immunohistochemistry. These observations indicate that microvascular endothelial cells of patients with SSc show abnormalities in a variety of genes that are able to account for defective angiogenesis.

Biopsy↗
PubMed2006

Domain 1 of the urokinase-type plasminogen activator receptor is required for its morphologic and functional, beta2 integrin-mediated connection with actin cytoskeleton in human microvascular endothelial cells: failure of association in systemic sclerosis endothelial cells.

OBJECTIVE: In systemic sclerosis (SSc) microvascular endothelial cells (MVECs), angiogenesis is blocked by matrix metalloproteinase 12-dependent cleavage of domain 1 of the urokinase-type plasminogen activator receptor (uPAR). Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and truncated uPAR are differentially associated with integrins and with motor components of the cytoskeleton. METHODS: SSc and normal MVECs were isolated from human skin biopsy specimens and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of truncated and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by reverse transcription-polymerasechain reaction. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting. RESULTS: Only full-size uPAR showed a connection with the actin cytoskeleton in ECs. This connection was mediated by the uPAR-associated alphaMu- and alphaX-subunits of beta2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with beta2 integrins or with actin. beta3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of Rac and Cdc42 (thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility) and blocked the integrin-engagement-delivered signals to the actin cytoskeleton. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that ligation of uPAR by uPA empowers the beta2/beta3 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc MVECs. CONCLUSION: The reduced angiogenic properties of SSc MVECs can be explained by the effects of uPAR truncation and the subsequent loss of the beta2 integrin-mediated connection of uPAR with the actin cytoskeleton in these ECs.

Actins↗
PubMed2006

Plasminogen activators and inhibitor type-1 in alveolar osteitis.

Alveolar osteitis (AO) is characterized by excess fibrinolysis, leading to early dissociation of the clot that normally follows tooth extraction. Nonetheless, scarce information is available on the fibrinolytic system in AO. In this study, we report on the differential composition of postextraction wound healing tissue and of peri-alveolar gingival epithelium from normal healing and AO patients in terms of plasminogen activators, plasminogen activator inhibitor-type 1, and urokinase-type plasminogen activator receptor. Plasminogen activators were studied by overlay zymography, western blotting, and enzyme-linked immunosorbent assay (ELISA). Plasminogen activator inhibitor-type 1 and urokinase receptor were measured by ELISA. In AO, the fibrinolytic activity of wound healing tissue was accounted for by an increase ( approximately 85%) of urokinase-type plasminogen activator, whereas tissue-type plasminogen activator was unchanged. Plasminogen activator inhibitor-type 1 showed a 6.7-fold increase in AO. These results point to key roles of urokinase in AO hyper-fibrinolysis and of plasminogen activator inhibitor type-1 in slowing down the healing response. Peri-alveolar gingival epithelium in AO showed an overall decrease of all the components of the fibrinolytic system, including the urokinase receptor, which indicates a decrease of the migration properties of epithelial cells.

Adult↗
PubMed2006

Cytokine-dependent invasiveness in B16 murine melanoma cells: role of uPA system and MMP-9.

Proteases are crucial for the spread of cancer cells from a primary tumor to the site of secondary growth. This study examined the ability of IFNgamma and TNFalpha to stimulate a better invasiveness in B16 murine melanoma cells, and investigated whether this enhanced ability was related to a higher expression of protease activities, such as urokinase plasminogen activator (uPA) and its receptor (uPAR), and matrix metalloproteinases 2 and 9 (MMP-2, MMP-9). We found that murine melanoma cells enhanced their lung-colonizing potential in vivo and invasiveness through Matrigel-coated filters upon costimulation with IFNgamma and TNFalpha; neither IFNgamma nor TNFalpha alone, at the dose used in the experiments, was able to elicit a change in the invasive/metastatic efficiency of melanoma cells. The invasive phenotype of murine melanoma cells stimulated with IFNgamma and TNFalpha was characterized by an enhanced uPA/uPAR and MMP-9 expression: TNFalpha promoted MMP-9 mRNA expression and pro-MMP-9 protein secretion, and the costimulation with IFNgamma and TNFalpha was required to potentiate the expression of mRNA and protein for uPAR, and to induce a redistribution of uPA from the soluble to the cell body-associated form. Both monoclonal antibodies, anti-uPAR and anti-MMP-9, caused a significant reduction of invasiveness in IFNgamma/TNFalpha-stimulated melanoma cells. These results indicate that invasiveness in B16 murine melanoma cells can be regulated in a cytokine-specific fashion and is dependent on the synergism between the uPA/uPAR system and MMP-9.

Animals↗
PubMed2005

The antiangiogenic tissue kallikrein pattern of endothelial cells in systemic sclerosis.

OBJECTIVE: Postnatal angiogenesis relies on a proper response of endothelial cells to angiogenic stimuli. In systemic sclerosis (SSc), endothelial cells are unresponsive to angiogenic factors. Since circumstantial and experimental evidence points to tissue kallikreins as powerful effectors of the angiogenic response, we undertook this study to investigate the kallikrein pattern of normal and SSc endothelial cells in order to identify differences that can account for defective angiogenesis. METHODS: Expression of 14 tissue kallikreins was studied by a microarray approach, by reverse transcription-polymerase chain reaction, and by Western blotting in endothelial cells isolated from the skin of clinically healthy subjects and SSc patients. Cell proliferation was quantified by direct cell counting. Invasion and capillary morphogenesis were evaluated in a Boyden chamber and in culture flasks layered with Matrigel. Cyclic nucleotide production was measured by enzyme immunoassay. MAP kinase and ERK activation were measured by Western blotting. RESULTS: Endothelial cells from SSc patients showed poor expression of kallikreins 9, 11, and 12 compared with endothelial cells from normal subjects. Antibodies against the relevant kallikreins on normal endothelial cells revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by normal cells in in vitro angiogenesis. Reduction of cAMP and cGMP production and of ERK phosphorylation upon administration of antikallikrein antibodies revealed that the activity of kallikreins 9, 11, and 12 was mediated by kinins. CONCLUSION: Reduction of tissue kallikreins 9, 11, and 12 may be relevant to reduced angiogenesis in SSc patients.

Antibodies, Blocking↗
PubMed2005

The urokinase-type plasminogen activator, its receptor and u-PA inhibitor type-1 affect in vitro growth and invasion of Kaposi's sarcoma and capillary endothelial cells: role of HIV-Tat protein.

The aggressive and malignant nature of AIDS-associated Kaposi's sarcoma (KS) lesions have largely been ascribed to Tat, the HIV-1 transactivator protein. Among other activities, HIV-Tat induces the migration and invasion of KS and endothelial cells. Since cell invasion is strictly correlated to the activity of lytic enzymes, we elucidated the role of the cell-associated plasminogen activation system in Tat-dependent and in constitutive invasion and proliferation of KS and of microvascular endothelial cells (MVEC). We demonstrate that KS cells and MVEC express the u-PA receptor (u-PAR) and release plasminogen activators and plasminogen activator inhibitor type-1 (PAI-1). The urokinase-type plasminogen activator (u-PA) is chemotactic, chemoinvasive and mitogenic for KS cells and for MVEC. Conditioned medium from KS cells induced invasion and proliferation of MVEC through the u-PA/u-PAR system. Tat is motogenic and mitogenic on KS cells and MVEC, and stimulates morphogenesis of MVEC. These activities were inhibited following antagonization of u-PA and u-PAR, which also reduced constitutive proliferation and invasion of KS cells and MVEC. These data indicate that the u-PA/u-PAR/PAI-1 system is involved in KS-induced endothelial cell invasion, proliferation, and differentiation. Further, exogenous Tat protein could up-regulate the fibrinolytic system, increasing its influence on KS and endothelial cell proliferation and migration, potentially promoting KS progression. These observations suggest the potential for application of u-PA/u-PAR system inhibitors for control of AIDS-associated KS, that has a high risk of recurrence with highly active antiretroviral therapy failure, and of other KS forms.

Antibodies, Monoclonal↗
PubMed2004

Antisense oligodeoxynucleotides for urokinase-plasminogen activator receptor have anti-invasive and anti-proliferative effects in vitro and inhibit spontaneous metastases of human melanoma in mice.

We have targeted the urokinase-type plasminogen activator receptor (uPAR) with phosphorothioate antisense oligonucleotides (aODN) in vitro to evaluate the anti-invasive and anti-proliferative effects of uPAR down-regulation, as well as in vivo to evaluate anti-tumor and anti-metastatic activity. aODN-dependent uPAR downregulation in vitro was induced in cells of human melanoma, mammary carcinoma, ovarian carcinoma and SV-40-transformed embryonic lung fibroblasts. uPAR was determined by an antibody-based assay and by semiquantitative polymerase chain reaction. Cell invasion was evaluated by Matrigel invasion assay and cell proliferation by direct cell counting. aODN reduced uPAR, invasion and proliferation in all the treated cell lines. Following aODN treatment, human melanoma cells exhibited a strong decrease of uPAR-dependent ERK1/2 activation and were used in vivo to control metastasis in CD-1 male nude (nu/nu) mice by uPAR aODN injection. 60 mice were injected in the hind leg muscles with a suspension of 10(6) melanoma cells. After 4 days, when a tumor mass of about 350 mg was evident in all the mice injected, 20 mice were treated i.v. with aODN and 20 with dODN at 0.5 mg/day for 5 consecutive days. Twenty control mice were not treated. A second and third cycle of treatment was administered at 2-day intervals. Treatment with aODN resulted into a 78% reduction of lung metastases and 45% reduction of the primary tumor mass with no loss of body weight. Our results suggest to evaluate the utility of uPAR aODN in controlling the metastatic spreading of human melanoma.

Animals↗
PubMed2004

Matrix metalloproteinase 12-dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis.

OBJECTIVE: Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell-associated urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process. METHODS: MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel-coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting. RESULTS: Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA-dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP-12. SSc MVEC-conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti-MMP-12 antibodies restored this SSc MVEC-induced impaired functioning. CONCLUSION: Overproduction of MMP-12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients.

Blotting, Western↗
PubMed2003

bcl-2 induction of urokinase plasminogen activator receptor expression in human cancer cells through Sp1 activation: involvement of ERK1/ERK2 activity.

We have previously demonstrated that Bcl-2 overexpression in human breast carcinoma and melanoma cells synergizes with hypoxia to increase angiogenesis through up-regulation of vascular endothelial growth factor. In this work we demonstrated, for the first time, that Bcl-2 overexpression in cancer cells exposed to hypoxia modulates urokinase plasminogen activator receptor (uPAR) expression through Sp1 transcription factor and that the extracellular signal-regulated kinase (ERK) pathway plays a role in Sp1 transcriptional activity. In particular, an increase in uPAR protein and mRNA expression was found in melanoma bcl-2 transfectants grown under hypoxia when compared with control cells, and a decrease of uPAR protein expression was induced by treatment of cells with specific bcl-2 antisense oligonucleotides. Up-regulation of uPAR expression was accompanied by increased Sp1 protein expression, stability, serine phosphorylation, and DNA binding activity. Treatment of cells with mitramycin A, an inhibitor of Sp1 activity, confirmed the role of Sp1 transcriptional activity in uPAR induction by Bcl-2. The contribution of the ERK pathway in Sp1-increased transcriptional activity was demonstrated by the use of chemical inhibition. In fact, ERK kinase activation was induced in Bcl-2-overexpressing cells exposed to hypoxia, and the ERK kinase inhibitor UO126 was able to down-regulate Sp1 phosphorylation and DNA binding activity. Using a human breast carcinoma line, we obtained data supporting our findings with melanoma cells and identified a link between the induction of Sp1 and uPAR expression as a common bcl-2-controlled phenomenon in human tumors. In conclusion, our results strongly indicate that up-regulation of uPAR expression by Bcl-2 in hypoxia is modulated by Sp1 DNA binding activity through the ERK signaling pathway.

Blotting, Northern↗
PubMed2002

Multiple pathways of cell invasion are regulated by multiple families of serine proteases.

The complex process of tumor invasion requires the coordinated expression and activity of cell-substratum adhesive interactions and of cell-associated protease systems, which destroy the extracellular matrix (ECM), in order to enable the invading cells to simultaneously grip and destroy the anatomical barriers that control cell spreading. A number of data indicate that such a 'grip and go' process may be performed by an enlarging series of cell membrane-associated serine proteases and serine protease receptors, which provide the invasive cells with a functional unit (the protease and its receptor), able to mediate cell-substratum adhesion through specific receptor domains, to proteolytically degrade ECM and to deliver into the cell signals that up-regulate the expression either of the protease/receptor complex, or of other adhesion molecules, such as integrins. There is evidence that some proteases and protease receptor expression are under the control of tumor hypoxia, which is the result of an imbalance in oxygen supply and demand. The urokinase-type plasminogen activator (u-PA) receptor (u-PAR) is under hypoxic control and cooperates with other serine proteases of the blood coagulation pathways that may extravasate in the tumor milieu as a result of hypoxia-simulated increase of vessel permeability. Other serine proteases and their receptors cooperate with the cell-associated fibrinolytic system to promote cell invasion. Among these, tissue factor and its ligand coagulation factor VII, thrombin and its protease-activated receptors, and type II trans-membrane serine proteases seem to play a crucial role. This Review takes into consideration the complex scenario of the single serine proteases and related receptors that are involved in cell invasion, as well as the protease receptor/adhesion molecule interplay which is necessary to focus the cell surface-driven proteolysis where adhesion provides a grip to the invading cell.

Animals↗
PubMed2002

Growth factor-dependent proliferation and invasion of muscle satellite cells require the cell-associated fibrinolytic system.

The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFbeta) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cell-associated fibrinolytic system. We have observed that the urokinase plasminogen activator (u-PA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly up-regulated by TGFbeta, mainly in dystrophic myoblasts. bFGF up-regulated u-PA in both normal and dystrophic myoblasts grown in primary culture, while a striking down-regulation was observed with TGFbeta. TGFbeta was the only growth factor able to exceptionally up-regulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factor-dependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cell-associated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of u-PA and of its receptor. Noteworthy, TGFbeta induced a dose-dependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFbeta-challenged dystrophic myoblasts undergo an exceptional up-regulation of the receptor and of PAI-1, we propose the possibility that the TGFbeta-induced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.

Case-Control Studies↗