Abstract
In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.
Highlights
Proteoglycans are a subset of complex polysaccharides bound covalently to a core protein in the extracellular matrix (ECM)
Since vascular endothelial growth factor 165 (VEGF165) was expressed in chorioallantoic membrane (CAM) [36], we hypothesized that the angiogenic activity of
We demonstrated that OTR4120, a member of a new pharmacological family called RGTA that mimics the function of natural GAGs, promoted angiogenesis via affinity for VEGF165
Summary
Materials—Dextran 40 USP was from Amersham Biosciences (Uppsala, Sweden), and heparin and collagen type I were from Sigma (Saint Quentin Fallavier, France). For the total RNA extraction, instead of in situ fixation, the CAM inside the ring was excised from five eggs for each sample tested, washed three times in phosphatebuffered saline (PBS), pH 7.4, and stored at Ϫ80 °C until use. The wells were washed three times with washing buffer, and the nonspecific interactions were saturated with PBS containing 3% BSA, for 1 h at room temperature. Saturation binding experiments were performed in the presence of VEGF165 at increasing concentrations (0 – 0.15 nM for VEGFR-1, 0 –1.2 nM for VEGFR-2, and 0 – 0.3 nM for NP-1) and of heparin or OTR4120 at 100 ng/ml, a concentration previously shown to induce maximal VEGF165 binding to its receptors. Lated goat anti-rabbit IgG (Chemicon International Inc., Temecula, CA) (1:1000 dilution) in saturation buffer, followed by three washes in PBS, 0.2% Tween 20 and incubation with an avidin-biotinylated-alkaline phosphatase complex (Vector Laboratories). The mounting medium for fluorescence was from Thermo Shandon (Pittsburgh)
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