11 research outputs found
GDF5 regulates TGFß-dependent angiogenesis in breast carcinoma MCF-7 cells: in vitro and in vivo control by anti-TGFß peptides
BACKGROUND: TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß. METHODS: We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis. RESULTS: TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis. CONCLUSIONS: TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies
Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.
Evaluation of ALK pattern and SMAD1/2/3/5 phosphorylation in MVECs.
<p><b>Panel A</b>. RT-PCR showing ALK expression on 3 of the MVEC cell lines used. GAPDH: loading control. Bp is shown on the right. The picture shows the results of a typical experiment out of 3 replicas. <b>Panel B.</b> Quantification of ALK RNA as related to GAPDH. The histogram shows the results obtained in 3 different experiments performed in duplicate on each MVEC cell line, ± SD. * p<0.05 (Student t-test), significantly different among various ALK RNAs. <b>Panel C</b>. Western blots of total protein extracts from MVECs exposed to CM of MCFM and MCFTß for 30 min and 120 min, respectively, in the presence or absence of P17. Western blots were probed for phospho-SMAD1/5, SMAD5, phospho-SMAD2, SMAD2/3 and Tubulin (loading control) as indicated. Molecular weights are reported on the right. Also in this case the picture shows the results of a typical experiment out of 3 replicas.</p
TGFß/GDF5-dependent angiogenesis in vitro and in vivo.
<p><b>Panel A</b>. Capillary morphogenesis experiments in control MVECs (untreated), upon MCFTß CM stimulation alone, and in the presence of irrelevant IgG (+ Irrel IgG), 5 µg/ml anti-GDF5 antibody (anti-GDF5), not-targeting siRNA (siNT), GDF5-targeting siRNA (siGDF5), 5 µg/ml anti-BM7 antibody (anti BMP7), BMP7-targeting siRNA (siBMP7). <b>Panel B</b>. Representative experiment of siGDF5-dependent and anti-GDF5 antibody-dependent inhibition of angiogenesis in the Matrigel sponge model in mice. <b>Panel C</b>. On the left: angiogenesis in the sponges was quantified by evaluating the haemoglobin content of each sponge. On the right: % vascularization ± SD in the same conditions. Images were acquired with the ZEISS SR Stemi stereomicroscope. TR: transfection reagents alone; siNT: not-targeting siRNA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050342#s3" target="_blank">Results</a> are the mean of three experiments (three animal for each condition, two Matrigel sponges in each animal). Graphs are shown as mean ± SD; * p<0.05 (Student t-test) with respect to both transfection reagents alone (TR) and not-targeting siRNA (siNT).</p
<i>In vitro</i> and <i>in vivo</i> evaluation of MCF7-TGFß-induced angiogenesis.
<p><b>Panel A</b>. MVECs were exposed for 6 hours to CM from MCFTß or MCFM. Numbers on the upper right side indicate the percent field occupancy of capillary plexus (±SD), taking untreated MVECs incubated in their own culture medium as 100%. Since the overall morphology did not substantially differ between 6 and 18 h after MVEC seeding (apart of a better definition of tubule formation by capillary projections), quantification was performed at 6 h after seeding. Pictures show a typical out of 4 different experiments that gave similar results. Dx: Doxycycline. The bottom pictures of panel A show capillary morphogenesis of MVECs exposed to CM of Dx−/MCFTß in the presence of p17 (+P17), p144 (+P144) or of a scrambled oligopeptide (+scr), respectively. * p<0.05 (Student t-test), significantly different from mock. <b>Panel B</b>. Upper part: Matrigel sponges, containing 2.5×10<sup>6</sup> MCFTß or MCFM, were recovered five days post implantation in 4 mice, each one grafted with 2 different sponges. The anti-angiogenesis activity of p17 peptide was evaluated in the presence of MCFTß. The figure shows a typical experiment out of 4 different experiments that gave similar results, upon systemic administration (i.p.) of the peptide. Co-injecting p17 in the plug with MCFTß gave similar results (not shown). Dx: Doxycycline. Lower part: CD31 immunohistochemistry of sponges in the same experimental conditions shown in the upper part. <b>Panel C</b>. On the left: haemoglobin contents ± SD versus MCFM +Dx taken as 100% in explanted specimes from 4 different experiments. On the right: % vascularization ± SD in the same conditions. * p<0.05 (Student t-test), significantly different versus the relevant control.</p
TGFß expression in transfected MCF7 cells. Effects of TGFß overproduction on MCF7 cells.
<p><b>Panel A</b>. Western blot of conditioned-media. MCFM: MCF7-Tet-Off transfected with the mock empty vector; MCFTß: MCF7-Tet-Off transfected with the mutated TGFß1-coding sequence. The figure reports a typical experiment out of 4 replicas. On the right, molecular weight expressed in kDa; Dx = Doxycycline. <b>Panel B</b>. ELISA kit determination of TGFß concentration in the culture medium of MCF7 in the same conditions as in panel A (4 different experiments performed in triplicate). * p<0.05 (Student t-test), significantly different from mock. <b>Panel C</b>. Confocal microscopy showing distribution of vimentin, E-cadherin and N-cadherin in MCFM and MCFTß. Original magnification: ×60. <b>Panel D</b>. Western blot of cell lysates. On the right, molecular weight expressed in kDa. Tubulin: loading control. The figures report a typical experiment out of 4 replicas. <b>Panel E</b>. Kinetics of % vital cells obtained by using the WST-1 reagent. Data are shown as the mean ±SD obtained in 4 different experiments performed in triplicate in the reported conditions. <b>Panel F</b>. MCFM and MCFTß cells Matrigel invasion in the Boyden chamber assay. Data are expressed as percent variations of invasive cells against the untreated control, taken as 100%, ±SD, obtained in 4 different experiments performed in triplicate. * p<0.05 (Student t-test), significantly different from mock. <b>Panel G</b>. Western blotting showing caspase activation. On the right, molecular weight expressed in kDa. Tubulin: loading control. The figure reports a typical experiment out of 4 replicas.</p
SMAD4 nuclear translocation.
<p>Numbers on the left indicate the elapsed time (hours) after addition of MCFM and MCFTß CM to MVEC. +P17: results obtained in the presence of the TGFß inhibiting peptide p17. The data show a typical experiment out of 3 replicas that gave similar results.</p