Cooperation between carboxy-terminal domains of VEGF-A dictates its biological properties in vitro and in vivo

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface Plasmon Resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparansulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exon 5 or exon 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b is angiostatic, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant

Etude d'un nouveau variant d'épissage alternatif de la Neuropilin-1: rôle antagoniste dans la régulation de la progression tumorale?

Neuropilin-1 (NRP1) is a transmembrane glycoprotein and a co-receptor for several growth factors, for example some variants of the Vascular Endothelial Growth Factor A (VEGF-A). It largely contributes to the regulation of angiogenesis but also to cancer formation. NRP1 can be considered as a proteoglycan as glycosaminoglycans side chains can be added on serine 612. Currently, six splice variants of NRP1 have been described. An additional form was recently identified in our laboratory. Depending upon the cell types, it represents 20-30% of the total amount of NRP1. As compared to the full size NRP1 (NRP1-FS), 7 amino acids are deleted. As the missing sequence is located 2 amino acids downstream of the Ser612 required for glycosaminoglycans addition, this process could be somehow affected and the function of the protein could be modified. The glycosylation of NRP1-FS and -Δ7 was analyzed in different cells overexpressing each isoform. Western blotting analyses suggested that NRP1-Δ7 was less glycosylated than NRP1-FS. Prostate cancer cells (PC3) were engineered to express NRP1-FS or –Δ7 only in the presence of doxycycline. The migration of these cells was analyzed by scratch assay, with or without doxycycline in the medium. As compared to controls and to NRP1-FS-expressing cells, production of NRP1-Δ7 was linked to a reduction of cell migration. A DNA dosage showed that NRP1-FS enhanced cell proliferation, while NRP1-Δ7 reduced it. Tumor growth was assessed in vitro by a culture in soft agar. As compared to control conditions, expression of NRP1-FS by doxycycline increased colonies formation. By contrast, NRP1-Δ7 inhibited colonies number, suggesting an inhibition of tumorigenesis by this variant. As PC3 cells express basal level of endogenous NRP1, this suggests some competitive inhibition of NRP1 functions by NRP1-Δ7. Finally, the function of each variant was investigated in vivo in a model of injection in the flanks of nude mice of PC3 cells conditionally expressing NRP1-FS or -Δ7. As compared to the control, NRP1-FS increased tumor size and weight. By sharp contrast, the expression of NRP1-Δ7 was associated with a reduction of tumorigenicity. Cells with forced expression of NRP1-Δ7 also developed fewer blood vessels as compared to the control. These results suggest that NRP1-Δ7 have an antagonistic action on cancer formation and angiogenesis.Study of the Neuropilin isoforms produced by alternative splicing: functional implications for the regulation of angiogenesis and tumor progressio

Un modèle murin de la surdominance polaire ?

peer reviewe

Synergy between dinotefuran and fipronil against the cat flea (Ctenocephalides felis): improved onset of action and residual speed of kill in adult cats

Abstract Background The cat flea, Ctenocephalides felis felis (C. felis), is a cosmopolitan hematophagous ectoparasite, and is considered to be the most prevalent flea species in both Europe and the USA. Clinical signs frequently associated with flea bites include pruritus, dermatitis and in severe cases even pyodermatitis and alopecia. Ctenocephalides felis is also a vector for several pathogens and is an intermediate host for the cestode Dipylidium caninum. Treatment of cats with a fast-acting pulicide, that is persistently effective in protecting the animal against re-infestation, is therefore imperative to their health. In addition, a rapid onset of activity (“speed of kill”) may also reduce the risks of disease transmission and flea allergic dermatitis. The aim of this study was to evaluate the in vitro insecticidal activity and potential synergism between dinotefuran and fipronil against C. felis. A further aim was to evaluate the onset of activity and residual speed of kill of the combination in vivo on cats artificially infested with C. felis. Methods In the first study, the insecticidal activity of dinotefuran and fipronil separately and dinotefuran/fipronil (DF) in combination, at a fixed ratio (2:1), was evaluated using an in vitro coated-vial bioassay. In the second study, the onset of activity against existing flea infestations and residual speed of kill of DF against artificial flea infestations on cats was assessed in vivo. Onset of activity against existing flea infestations was assessed in terms of knock-down effect within 2 h post-treatment and onset of speed of kill assessed at 3 h, 6 h and 12 h post-treatment. Residual speed of kill was evaluated 6 h and 48 h after infestation, over a period of six weeks post-treatment. Results In vitro results revealed that the DF combination was synergistic and more potent against fleas than either compound alone. The combination also proved effective when tested in vivo. Efficacy was > 97% [geometric mean (GM) and arithmetic mean (AM)] at 3 h after treatment, and ≥ 99.8% (GM and AM) at 6 h and 12 h post-treatment. At 6 h after flea re-infestations, the efficacy of DF remained ≥ 90.8% (GM and AM) for up to 28 days, and at 42 days post-treatment persistent efficacy was still ≥ 54.3% (GM and AM). At 48 h after flea re-infestations, DF remained almost fully effective for up to 28 days, with efficacies ≥ 99.4% (GM and AM) and was persistently ≥ 93.0% (GM and AM) effective for up to 42 days post-treatment. Conclusions The combination of dinotefuran and fipronil in a single formulation exhibited strong synergistic insecticidal activity against C. felis in vitro, and also proved effective on artificially infested cats. This activity had a rapid onset that persisted for 6 weeks against re-infestations of C. felis on cats. The rapid curative insecticidal effect was observed as early as 3 h after treatment, and as early as 6 h after re-infestations for up to 6 weeks post-treatment. The insecticidal activity profile of DF makes it an optimal candidate for the protection of cats against flea infestations, and possibly also associated diseases

Evaluation of the use of VEGF111 for the treatment of tendon lesions.

Alterations of tendons are common pathologies resulting from repetitive or abnormal mechanical sollicitations. Very frequently lesions become chronic and may even lead to rupture. As there is no current efficient treatment for curing this type of diseases, new therapeutic approaches are being tested and developed. Injection of platelet-rich plasma (PRP) seems to be a promising treatment by local release of growth factors. Among these factors, VEGF-A is known to induce positive effects on vascular functions and angiogenesis, and could be implicated in the healing process of tendons. Several isoforms of VEGF-A have been described in literature, including VEGF165 and 121. VEGF111 is encoded by exons 1-4 and 8a. The lack of exon 5 enables VEGF111 to resist to proteolytic degradation and the absence of exons 6 and 7 reduces its affinity for several macromolecules present on the cell surface and in the extracellular matrix. In vivo, it has been shown to be highly proangiogenic and diffusible. A 5mm defect was surgically performed in the Achilles tendon of 60 rats. Two hours after closure of the fascia and the skin, an injection within the wound was performed with PBS alone (n=30) or with PBS containing 100 ng of VEGF111 (n=30). 10 rats of each group were sacrificed at days 5, 15 and 30. The operated tendon was then carefully removed and collected for either immunohistochemical analyses or mechanical testing. At each time point, the section and the overall appearance of the repairing tendons were similar for PBS and VEGF111-injected tendon. As compared to controls, injection of VEGF111 seemed to promote a faster angiogenesis, although the number of samples was at this stage too low for performing reliable statistical analysis. Mechanical resistance to rupture of the repairing tendons was also measured. No difference between the two groups was observed after 5 or 15 days. By contrast, increased tensile strength was clearly evidenced in the VEGF-treated group after 30 days. These preliminary data seem to indicate a positive effect of a single VEGF111 injection for restoring the mechanical properties of tendons after their section. Additional experiments are planned for confirmation purposes and for further characterizing the model. It includes a “dose- response” analysis, the use of VEGF165 as an additional control and a study evaluating the effect of several injections

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF(111)a, VEGF(111)b, VEGF(121)a, VEGF(121)b, VEGF(155)a, VEGF(155)b, VEGF(165)a, VEGF(165)b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF(165)b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF(111)b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant