1,721,020 research outputs found
Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2.
No full textDownstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK(1/2) activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK(1/2) phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel. Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK(1/2) in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27,30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK(1/2)-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant Δ27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK(1/2) from its ability to stimulate uPA upregulation and/or in vitro angiogenesis. In conclusion, the data indicate that ERK(1/2) phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK(1/2) activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells
The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate proteoglycans: biochemical bases and biological implications
No full textImpact of fibroblast growth factor-2 (FGF2) on tumor growth and vascularization: Establishing a "FGF2-dependent" tumor model
No full textLong Pentraxin-3 Inhibits FGF8b-Dependent Angiogenesis and Growth of Steroid Hormone-Regulated Tumors.
No full textFibroblast growth factor-8b (FGF8b) exerts nonredundant autocrine/paracrine functions in steroid hormone-regulated tumors. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we assessed the capacity of PTX3 to antagonize FGF8b and to inhibit the vascularization and growth of steroid hormone-regulated tumors. Surface plasmon resonance analysis shows that PTX3 binds FGF8b with high affinity (K d = 30-90 nmol/L). As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits dihydrotestosterone (DHT)- and FGF8b-driven proliferation of androgen-regulated Shionogi 115 (S115) mouse breast tumor cells. Furthermore, DHT-treated, PTX3 overexpressing hPTX3-S115 cell transfectants show a reduced proliferation rate in vitro and a limited angiogenic activity in the chick embryo chorioallantoic membrane and murine s.c. Matrigel plug assays. Accordingly, hPTX3-S115 cells show a dramatic decrease of their tumorigenic activity when grafted in immunodeficient male mice. These results identify PTX3 as a novel FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of hormonal tumor
Biochemical bases of the interaction of the human basic fibroblast growth factor with glycosaminoglycans: new insights from trypsin digestion studies
No full textHeparins from bovine mucosa and lung, and chemically modified
heparins were assayed for their capacity to: (i) protect human
recombinant basic fibroblast growth factor (bFGF) from tryptic
cleavage; (ii) prevent 1251-bFGF binding to heparan sulphate
proteoglycans present in the extracellular matrix and on the cell
surface of fetal bovine aortic endothelial GM 7373 cell cultures;
(iii) affect 1251-bFGF binding to high-affinity tyrosine kinase
FGF receptors present on the cell membrane of GM 7373 cells;
(iv) inhibit the mitogenic activity exerted by bFGF in the same
cells. The results demonstrate thatthe potency shown by mucosal
heparins in the different assays is a direct function of size, verylow-
molecular-mass heparin (2.0 kDa) being significantly less
effective on a molar basis than unfractionated heparin (13.6 kDa).
Increased flexibility of the backbone structure, as observed in
reduced/oxidized heparins of different size, does not affect the
capacity of the polysaccharide to interact with bFGF. In contrast,
selective 2-O-desulphation, but not 6-O-desulphation, drastically
reduced the capacity of heparin to protect bFGF from proteolytic
cleavage, to affect its interaction with low- and high-affinity sites,
and to inhibit its mitogenic activity. Two preparations of bovine
lung heparin, differing in molecular mass, were as effective as
mucosal heparin in the bFGF-tryptic-digestion assay and the
endothelial-cell proteoglycan-binding assay, but they were highly
inefficient at inhibiting the capacity of bFGF to interact with its
tyrosine kinase receptors. Bovine lung heparins were also less
effective than mucosal heparin as bFGF antagonists in GM
7373-cell-proliferation assays. N-Desulphated/N-acetylated
bovine lung heparin retained only a significant capacity to
protect bFGF from tryptic cleavage. The results demonstrate
that different chemical features of the heparin molecule, including
decrease in molecular mass, selective desulphation, disaccharide
composition and clustering, affect differently the capacity of the
glycosaminoglycan to interact with bFGF and to influence its
biological behaviour in different assays in vitro and in endothelial
cell cultures. Our findings should aid the design of synthetic
oligosaccharides aimed at improving the. bioavailability of bFGF
when administered in vivo as a therapeutic agent
Distinct role of 2-O, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1
No full textInteraction of basic fibroblast growth factor (bFGF) with heparan sulfate proteoglycans (HSPGs) plays an important role in the binding of bFGF to its tyrosine kinase receptor (FGFR). The molecular bases of this interaction were investigated by evaluating the capacity of conventional and selectively desulfated heparins i) to affect the binding of bFGF to FGFR and HSPGs of NIH 3T3 cells transfected with FGFR-1/flg cDNA, ii) to facilitate the interaction of bFGF with a recombinant soluble form of the extracellular domain of FGFR-1/flg (xcFGFR-1), and iii) to protect xcFGFR-1 from tryptic cleavage. 6-O-desulfated (6-O-DS) heparin, but not 2-O-desulfated (2-O-DS) and N-desulfated/N-acetylated (N-DS/N-Ac) heparins, retains the capacity to bind bFGF, as assessed by its ability to inhibit bFGF-binding to cell-associated FGFR-1 and HSPGs. On the other hand, at variance with conventional heparin, 2-O-DS, N-DS/N-Ac, and 6-O-DS heparins are all ineffective in potentiating the binding of bFGF to xcFGFR-1 and protecting xcFGFR-1 from tryptic cleavage. The data indicate that 6-O-sulfate groups are not essential for the interaction of heparin with bFGF but are involved in the interaction with xcFGFR-1. Our findings support the hypothesis that HSPGs modulate the binding of bFGF to FGFR through the formation of a ternary complex in which the glycosaminoglycan chains interact with bFGF via 2-O- and N-sulfate groups and with FGFR also via 6-O-sulfate groups
Stromal delivery of long Pentraxin-3 impairs FGF/FGFR-dependent tumor growth and metastasis
No full textThe FGF/FGFR system contributes to cancer progression by inducing tumor growth and neovascularization, thus representing an emerging therapeutic target. Long Pentraxin-3 (PTX3) is a soluble pattern recognition receptor expressed by endothelial and immune cells in inflammatory contexts. Among various ligands, PTX3 binds different members of the FGF family, acting as a natural FGF ligand trap.
Here, we generated transgenic mice expressing human (h)PTX3 under the control of endothelial specific Tie2/Tek transcription regulatory sequences (Tie2-hPTX3 mice). These animals accumulate significant levels of hPTX3 in perivascular stroma and in the blood stream. On this basis, Tie2-hPTX3 mice were used to investigate the impact of stroma delivery of hPTX3 on tumor growth, vascularization and metastasis.
The anti-angiogenic activity of endothelium-derived hPTX3 was confirmed by ex vivo aorta ring and in vivo matrigel plug assays. Next, different syngeneic FGF-dependent tumor cell lines, including TRAMP-C2 prostate carcinoma, B16-F10 melanoma and Lewis Lung carcinoma cells, were subcutaneously injected in Tie2-hPTX3 mice. Notably, the growth of all tumor grafts was significantly reduced in Tie2-hPTX3 mice when compared to wild type animals and was accompanied by a significant reduction of FGFR1 phosphorylation, decrease of tumor vascularity and tumor cell proliferation. Also, B16-F10 melanoma and M5076 ovarian sarcoma cells showed a dramatic decrease of their capacity to form experimental metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. Also, the orthotopic growth of syngeneic pancreatic and mammary tumor cells was significantly reduced after injection in Tie2-hPTX3 mice and led to increased survival compared to control mice. Finally, double transgenic TRAMP/Tie2-hPTX3 mice showed a significant delay of multistage prostate tumor onset and progression in respect to TRAMP mice.
Our findings demonstrate for the first time that in vivo delivery of PTX3 exerts a dramatic impact on tumor growth, vascularization and metastasis. These results have set the basis for the identification of a low molecular weight PTX3-derived molecule that recapitulates the FGF-trap activities of PTX3 and exhibits promising therapeutic potential for FGF-dependent tumors
Fibroblast growth factor-2 antagonist and Antiangiogenic activity of long-pentraxin 3-derived synthetic peptides
No full textAbstract: Angiogenesis and inflammation are closely integrated processes. Fibroblast growth factor-2 (FGF2) is a
prototypic angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 exerts its proangiogenic
activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors,
heparan-sulfate proteoglycans, and integrins. A tight cross-talk exists between FGF2 and the inflammatory response in the
modulation of blood vessel growth. Pentraxins act as soluble pattern recognition receptors with a wide range of functions
in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with shortpentraxins
and possesses a unique N-terminal domain. These structural features indicate that PTX3 may have distinct
biological/ligand recognition properties when compared to short-pentraxins. Co-expression of PTX3 and FGF2 has been
observed in different inflammation/angiogenesis-dependent diseases. PTX3 binds FGF2 with high affinity and specificity.
The interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the
angiogenic activity of the growth factor. This suggests that PTX3 may exert a modulatory function by limiting the
angiogenic activity of FGF2. An integrated approach that utilized PTX3 fragments, monoclonal antibodies, and surface
plasmon resonance analysis has identified the FGF2-binding domain in the unique N-terminal extension of PTX3. On this
basis, PTX3-derived synthetic peptides have been designed endowed with a significant antiangiogenic activity in vitro and
in vivo. They may provide the basis for the development of novel antiangiogenic FGF2 antagonists
Impact of fibroblast growth factor-2 on tumor microvascular architecture. A tridimensional morphometric study.
No full textThree cell clones originated by transfection of human endometrial adenocarcinoma HEC-1-B cells with fibroblast growth factor-2 (FGF-2) cDNA and characterized by a different capacity to produce and secrete the growth factor were transplanted subcutaneously in nude mice. Corrosion casting of the tumor microvasculature of xenografts produced by injection of 2 x 106 or 10 x 106 FGF-2-B9 cells (which produce and secrete significant amounts of FGF-2), 10 x 106 FGF-2-A8 cells (which produce comparable amounts of FGF-2 but do not secrete it), or 10 x 106 control FGF-2-B8 cells (which produce only trace amounts of FGF-2) was performed after 14 days of growth. Inter- branching distances, intervascular distances, branching angles, and vessel diameters were then determined using tridimensional stereo pairs of the casted tumor vascularity. When transplanted at the same concentration, FGF- 2-B9 cells grew faster in nude mice compared with FGF-2-A8 and FGF-2-B8 clones. The total amount of new vessel formation was far higher in FGF-2-B9 tumors than in FGF-2-B8 or FGF-2-A8 tumors. Also, vessel courses were more irregular and blind-ending vessels and evasates were more frequent in FGF-2- B9 tumors. Moreover, FGF-2-B9 tumor microvasculature was characterized by a wider average vascular diameter and by an extreme variability of the diameter of each individual vessel along its course between two ramifications. No statistical differences were observed when the distribution curves of the values of intervascular distances, interbranching distances, and branching angles of the microvessel network were compared among the different experimental groups. The distinctive features of the micro- vasculature of FGF-2-B9 tumors were retained, at least in part, in the smaller lesions produced by injection of a limited number of cells. The data indicate that FGF-2 production and release confer to FGF-2-B9 cells the ability to stimulate the formation of new blood vessels with distinctive architectural features. Neovascularization of FGF-2-B9 lesions parallels the faster rate of growth of the neoplastic parenchyma. This does not affect the overall architecture of the microvessel network that appears to be primed by characteristics of the HEC-1-B tumor cell line and/or by the microenvironment of the host. To our knowledge, this work represents the first attempt to define the influence of a single, defined growth factor on the tridimensional tumor vascular pattern
Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR
No full textThe subcutaneous Matrigel plug assay in mice
is a method of choice for the in vivo evaluation of pro- and
anti-angiogenic molecules. However, quantification of the
angiogenic response in the plug remains a problematic task.
Here we report a simple, rapid, unbiased and reverse
transcription-quantitative PCR (RT-qPCR) method to
investigate the angiogenic process occurring in the Matrigel
plug in response to fibroblast growth factor-2 (FGF2).
To this purpose, a fixed amount of human cells were added
to harvested plugs at the end of the in vivo experimentation
as an external cell tracer. Then, mRNA levels of the panendothelial
cell markers murine CD31 and vascular
endothelial-cadherin were measured by species-specific
RT-qPCR analysis of the total RNA and data were normalized
for human GAPDH or b-actin mRNA levels. RTqPCR
was used also to measure the levels of expression in
the plug of various angiogenesis/inflammation-related
genes. The procedure allows the simultaneous, quantitative
evaluation of the newly-formed endothelium and of nonendothelial/
inflammatory components of the cellular infiltrate
in the Matrigel implant, as well as the expression of
genes involved in the modulation of the angiogenesis
process. Also, the method consents the quantitative
assessment of the effect of local or systemic administration
of anti-angiogenic compounds on the neovascular response
triggered by FGF
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