Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.
ABSTRACT To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions.
Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4.
Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia.
The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.
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ABSTRACT: Retinal neovascularisation occurs under the influence of angiogenic factors that are induced by hypoxia, like vascular endothelial growth factor (VEGF), which is one of the major mediators. PTK/ZK inhibits VEGF signal transduction by blocking the tyrosine kinase of all three VEGF receptors. PTK/ZK is currently being evaluated in clinical trials for angioinhibitory therapy in tumour patients. To avoid potential systemic side effects, local application would be desirable for the treatment of ischemic retinopathies in humans. We therefore investigated the effect of intravitreally applied PTK/ZK in a mouse model for ischemia-induced retinopathy. C57BL/6J mice were placed in 75% oxygen on postnatal day 7. On day 12, they were treated with an intravitreal injection of PTK/ZK (5 microM or 40 microM) in one eye and buffer solution in the fellow eye. Afterwards, the animals were kept in room air until intracardial perfusion with fluorescein-dextran on day 17. Retinal whole mounts were prepared and ischemic retinopathy was evaluated using a standardised retinopathy score. A single intravitreal injection of 40 microM PTK/ZK reduced angioproliferative changes compared to the control eye of each animal (n=37). The difference in retinopathy scores was highly significant (P=0.002, Wilcoxon signed-rank test). Injection of 5 microM PTK/ZK did not show a significant antiangiogenic effect. Tyrosine kinase inhibitors are promising substances not only in cancer therapy, but also in the treatment of ischemic retinopathies that are mediated by VEGF. We showed that in a mouse model for ischemia-induced retinopathy a single intravitreal injection of 40 microM PTK/ZK is capable of significantly reducing angioproliferative retinopathy. The local application of PTK/ZK could be a new way to treat ischemic ocular diseases such as diabetic retinopathy in humans.Albrecht von Graæes Archiv für Ophthalmologie 07/2005; 243(6):593-600. · 1.93 Impact Factor
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ABSTRACT: Contact-dependent interactions between endothelial cells (ECs), as well as between ECs and mural cells, play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have identified ephrinB2 and EphB4 as markers and makers of arteriovenous differentiation during vascular development. On the basis of these findings, we hypothesized that Ephephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions that provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts anti-adhesively and induces detachment of ECs, as demonstrated by (1) inhibition of adhesion to ephrinB2-Fc-coated culture dishes, (2) detachment of ECs grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, vascular endothelial growth factor (VEGF) gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is pro-adhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2-mediated repulsive signals are transduced by EphB4, as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4-overexpressing ECs. Confrontation experiments of EphB4-overexpressing ECs with ECs overexpressing full-length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, these data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.Journal of Cell Science 07/2003; 116(Pt 12):2461-70. · 5.88 Impact Factor
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ABSTRACT: To develop oxygen-induced retinopathy in the mouse with reproducible and quantifiable proliferative retinal neovascularization suitable for examining pathogenesis and therapeutic intervention for retinal neovascularization in retinopathy of prematurity (ROP) and other vasculopathologies. One-week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then to room air. A novel fluorescein-dextran perfusion method has been developed to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in 6 microns sagittal cross-sections. Cross-sections were also stained for glial fibrillary acidic protein (GFAP). Fluorescein-dextran angiography delineated the entire vascular pattern, including neovascular tufts in flat-mounted retinas. Hyperoxia-induced neovascularization occurred at the junction between the vascularized and avascular retina in the mid-periphery. Retinal neovascularization occurred in all the pups between postnatal day 17 and postnatal day 21. There was a mean of 89 neovascular nuclei per cross-section of 9 eyes in hyperoxia compared to less than 1 nucleus per cross-section of 8 eyes in the normoxia control (P < 0.0001). Proliferative vessels were not associated with GFAP-positive astrocyte processes. The authors have described a reproducible and quantifiable mouse model of oxygen-induced retinal neovascularization that should prove useful for the study of pathogenesis of retinal neovascularization as well as for the study of medical intervention for ROP and other retinal angiopathies.Investigative Ophthalmology & Visual Science 01/1994; 35(1):101-11. · 3.44 Impact Factor