FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells.

BACKGROUND: Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. RESULTS: By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. CONCLUSIONS: We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

Design of PEGylated Three Ligands Silica Nanoparticles for Multi-Receptor Targeting.

The synthesis of silica nanoparticles (SiNPs) decorated on their surface with a range of various elements (e.g., ligands, drugs, fluorophores, vectors, etc.) in a controlled ratio remains a big challenge. We have previously developed an efficient strategy to obtain in one-step, well-defined multifunctional fluorescent SiNPs displaying fluorophores and two peptides ligands as targeting elements, allowing selective detection of cancer cells. In this paper, we demonstrate that additional level of controlled multifunctionality can be achieved, getting even closer to the original concept of "magic bullet", using solely sol-gel chemistry to achieve conjugation of PEG chains for stealth, along with three different ligands. In addition, we have answered the recurrent question of the surface ungrafting by investigating the stability of different siloxane linkages with the ERETIC Method (Electronic Reference to Access In Vivo Concentrations) by 19F NMR quantification. We also compared the efficiency of the hybrid silylated fluorophore covalent linkage in the core of the SiNP to conventional methods. Finally, the tumor-cell-targeting efficiency of these multi-ligand NPs on human endothelial cells (HUVEC or HDMEC) and mixed spheroids of human melanoma cells and HUVEC displaying different types of receptors were evaluated in vitro.

The presence of PEG on nanoparticles presenting the c[RGDfK]- and/or ATWLPPR peptides deeply affects the RTKs-AKT-GSK3ß-eNOS signaling pathway and endothelial cells survival.

Covering the surface of a nanoparticle with polyethylene glycol (PEG) is a common way to prevent non-specific interactions but how its presence impacts on the activity of targeting ligands is still poorly documented. We synthesized a set of 9 silica nanoparticles grafted with c[RGDfK]-, a peptide targeting integrin αvß3 (cRGD), and/or with ATWLPPR, an anti-neuropilin 1 peptide (ATW). We then added various PEGs, and studied NPs binding on primary endothelial cells, the downstream activated signaling pathways and the impact on apoptosis. Our results show that the presence of PEG2000 on cRGD/ATW nanoparticles moderately improves cell binding but induces a 6000 times augmentation of AKT-dependent cell response due to the recruitment of other Receptor Tyrosine Kinases. Augmenting the length of the spacer that separates the peptides from the silica (using PEG3000) mainly resulted in a loss of specificity. Finally, the PEG-mediated hyperactivation of AKT did not protect endothelial cell from dying in the absence of serum, while its moderate activation obtained without PEG did. Finally, PEGylation of cRGD/ATW-NPs can generate nanoparticles with potent capacities to activate the AKT-GSK3ß-eNOS cascade and to affect the resistance of endothelial cells to apoptosis. Thus, the impact of PEGylation should be precisely considered in order to avoid the apparition of counter-productive biological responses.

Evaluation of the theranostic properties of gadolinium-based nanoparticles for head and neck cancer.

BACKGROUND: The aim of the study was to evaluate the benefits of the combination of Gadolinium-based nanoparticles AGuIX and radiotherapy on the recurrence free survival after tumor resection in a head and neck animal orthotopic model. METHODS: Human head and neck CAL33 orthotopic tumors were implanted in female NMRI nude mice. The biodistribution of AGuIX was studied by fluorescence imaging. Tumor resection was performed 19 days after tumor implantation. Radiotherapy was performed 23 days after resection (10 Gy), 1 hour after AGuIX IV injection. RESULTS: After systemic administration, AGuIX passively accumulated in the orthotopic tumors. After tumor surgery, the combination of AGuIX with radiotherapy significantly improved the recurrence free survival and the median survival time (196 days) compared to irradiated only mice (75 days). CONCLUSION: This study demonstrated the improvement of the recurrence free survival following combination of AGuIX injection with radiotherapy after Head and neck tumor resection.

The sVEGFR1-i13 splice variant regulates a ß1 integrin/VEGFR autocrine loop involved in the progression and the response to anti-angiogenic therapies of squamous cell lung carcinoma.

BACKGROUND: While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known. METHODS: mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples. RESULTS: We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/ß1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and ß1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC. CONCLUSIONS: Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies.

"Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing αvß3 Integrins.

A new class of "polymultivalent" ligands combining several ligand clusters and a water-soluble biocompatible polymer is introduced. These original conjugates bear two levels of multivalency. They are prepared by covalent coupling of a controlled number of tetrameric cRGD peptide clusters along a well-defined copolymer synthesized by RAFT polymerization. The presence of multiple copies of peptide clusters on the same polymer backbone resulted in a much-higher relative potency than the free cluster reference. Thanks to the "polymultivalency", up to ∼2 orders of magnitude potency enhancement was reached in a competitive cell adhesion assay (nanomolar-range IC50 values). In addition, confocal microscopy and flow cytometry demonstrated that fluorescent "polymultivalent" conjugates (emitting in the far-red/near-infrared region) were able to specifically and selectively label cells expressing αvß3-integrin, the natural receptor of cRGD.