PO-44 - Tissue factor pathway inhibitor-2 (TFPI-2) is cleaved by PRSS3: implication for tumor endothelial cells migration.

INTRODUCTION: Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor whose expression is up-regulated by VEGF in microvascular and umbilical vein endothelial cells (EC). Despite this, TFPI-2 has been suggested as anti-angiogenic molecule, due to its ability to inhibit the migration/proliferation of EC induced by VEGF. Nothing is known about the precise mechanism of TFPI-2 function tuning in tumor endothelium. AIM: Aim of this study was to investigate the role of TFPI-2 in tumor vasculature, where angiogenesis and vascular remodeling are fundamental for cancer progression. MATERIALS AND METHODS: Tumor-EC were isolated from ovarian carcinomas and cultured in vitro in presence of factors reproducing the tumor microenvironment (VEGF, FGF-2, EGF). TFPI-2 and PRSS3 silencing was achieved by small interfering RNA (siRNA). Tumor-EC migration was assayed by the wound healing assay. Transcript expression was examined by qRT-PCR. Proteolytic reactions were monitored by western blot. RESULTS: We show that tumor-EC express TFPI-2, the majority of which is released and found anchored in the extracellular matrix. Silencing the expression of TFPI-2 enhances tumor-EC migration, confirming TFPI-2 as an anti-angiogenic molecule. We had previously shown that the cancer vasculature express PRSS3, a trypsin family member able to cleave proteins containing the kunitz-type domains; we reasoned that it could potentially inhibit TFPI-2. Herein, we demonstrate in a cell free system that TFPI-2 directly interacts with and is degraded by active PRSS3. In a more complex biological context, active PRSS3 is able to remove TFPI-2 from the extracellular matrix put down by tumor-EC. Accordingly, silencing PRSS3 causes the extracellular accumulation of TFPI-2 that results in the inhibition of tumor-EC migration. CONCLUSIONS: Our results demonstrate for the first time that TFPI-2 is a direct substrate of PRSS3, which hydrolyses TFPI-2 (most likely at the Kunitz-type domains) blocking its anti migratory capability. The proteolytic inactivation of TFPI-2 by PRSS3 might represent a mechanism favoring cancer by increasing angiogenesis and vascular remodeling. ACKNOWLEDGEMENT: Supported by the Italian Association for Cancer Research (AIRC).

Speciation study of the anti-inflammatory drug tenoxicam (Htenox) with Cu(II): X-ray crystal structure of [Cu(tenox)(2)(py)(2)].EtOH.

A speciation study was carried out in aqueous solution of the anti-inflammatory drug tenoxicam (Htenox), under quasi-physiological conditions (temperature of 37 degrees C and ionic strength 0.15 M NaCl) in order to determine the acidity constants from spectrophotometric studies, the pK(a) values found being pK(1)=1.143+/-0.008 and pK(2)=4.970+/-0.004. Subsequently, the spectrophotometrical speciation of the different complexes of Cu(II) with the drug was performed under the same conditions of temperature and ionic strength, observing the formation of Cu(Htenox)(2)(2+) with log beta(212)=20.05+/-0.01, Cu(tenox)(2) with log beta(012)=13.6+/-0.1, Cu(Htenox)(2+) with log beta(111)=10.52+/-0.08, as well as Cu(tenox)(+) with log beta(011)=7.0+/-0.2, all of them in solution, and solid species Cu(tenox)(2)(s) with an estimated value of log beta(012)(s) approximately 18.7. The crystalline structure of the complex [Cu(tenox)(2)(py)(2)]. EtOH, was also determined, and it was observed that tenoxicam employs the oxygen of the amide group and the pyridyl nitrogen to bond to the cation.

p73 Overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis.

Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.

[Serous cystadenoma of the pancreas: imaging findings]. / Cistoadenoma sieroso del pancreas: i rilievi del'imaging.

PURPOSE: To present the imaging findings of serous cystadenoma (SCA) and discuss the main problems of differential diagnosis with other cystic pancreatic lesions. MATERIALS AND METHODS: 55 cases of SCA were reviewed; all the lesions proved to be benign at pathology. 35 tumors were evaluated with Ultrasonography (US), 49 with Computed Tomography (CT), and 15 with Magnetic Resonance Imaging (MRI). RESULTS: Among the patients who underwent either demolitive (47) or derivative (3) interventions, two different morpho-structural patterns were identified: microcystic (37), and oligocystic (13). The diagnosis of SCA, possible in the presence of microcystic pattern, was achieved in 63% of cases by US (22/35), in 63% of cases by CT (31/49), and in 73% of cases by MRI (11/15). In 12 patients evaluated with all the imaging modalities, the combined information allowed a correct diagnosis in 10 cases (83%). The 13 oligocystic tumors were almost always undistinguishable from other cystic masses of the pancreas. CONCLUSIONS: The diagnosis of SCA can be considered certain if a microcystic pattern is detected. A correct diagnosis is not achievable in the presence of oligocystic architecture. MRI is the best imaging modality in characterizing this tumor.

Beryllium(II) complexes of the Kläui tripodal ligand cyclopentadienyltris(diethylphosphito-p)cobaltate(-).

The interactions of the beryllium(II) ion with the cyclopentadienyltris(diethylphosphito-P)cobaltate monoanion, L(-), have been investigated, in aqueous solution, by synthetic methods, potentiometry, ESMS, and (1)H, (31)P, and (9)Be NMR spectroscopy. L(-) has been found able to displace either two or three water molecules in the beryllium(II) coordination sphere, to form mononuclear, dinuclear, and trinuclear derivatives, in which the metal ion is pseudotetrahedrally coordinated. The species [BeL(H(2)O)](+) and [Be(2)L(2)(mu-OH)](+) have been identified in solution while complexes of formula BeL(2) and [Be(3)L(4)](ClO(4))(2) have been isolated as solid materials. The species [BeL(OPPh(2))](+), closely related to [BeL(H(2)O)](+), has been characterized in acetone solution and isolated as tetraphenylborate salt. The structure of the unusual trimeric complex [Be(3)L(4)](2+) has been elucidated by an unprecedented 2D (9)Be-(31)P NMR correlation spectrum showing the presence of a single central beryllium nucleus and two equivalent terminal beryllium nuclei. The three beryllium centers are held together by four cobaltate ligands, which display two different bonding modes: two ligands are terminally linked with all the three oxygen donors to one terminal beryllium, and the other two bridge two metal centers, sharing the oxygen donors between central and terminal beryllium atoms.

An approach to nasal tip remodeling.

The luxation (cartilage delivery) technique may be used to achieve a predictable, individualized result in nasal tip rhinoplasty. The author describes how this technique, using a bipedicle flap with an intercartilaginous and rim incision, allows superior visualization of the domal region without structural disruption of the cartilaginous arch. (Aesthetic Surg J 2001;21:345-348.).

Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells.

Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24. 24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis.