VEGF and neuropeptides in experimental choroidal neovascularization

Wet age-related macular degeneration (ARMD) is the most frequent reason for vision loss in elderly patients in the civilized world and is characterized by the formation of new vessels (CNVs) which sprout from the choroid into the subretinal space. The pathogenesis of this new vessel formation is not really clear but vascular endothelial growth factor (VEGF) plays a significant role since the therapy with VEGF-antibodies is well established in wet ARMD. However, it is not clear to which extent VEGF contributes to the pathogenesis and whether the neuropeptides substance P (SP), secretoneurin (SN) and neuropeptide Y (NPY) are also involved which act in a proangiogenic manner with a potency similar to that of VEGF. Subsequently, this project proposal intends to evaluate to which extent VEGF and the neuropeptides SP, SN and NPY contribute to the pathogenesis of CNVs using an appropriate animal model, in particular laser-induced CNVs in mice. For this purpose both in vitro as well as in vivo studies are performed. In vitro, the receptor-mediated effect of the tachykinins, SN and NPY on the migration and proliferation of human choroidal endothelial cells is investigated as well as their potency to induce capillary tube formation. In vivo, formation of CNVs after laser-induction is quantitatively evaluated by Spectral- Domain Optical Coherence Tomography and also ex vivo on choroidal flat mounts. Firstly, it is explored whether the size of CNVs is reduced in Y2-, NK1- and SN-k.o. mice. Then, the efficacy of an intravitreal injection of a SN- antibody, a nonpeptide Y2 antagonist (BIIE 0246) and a NK1 antagonist (SR140333) should be explored in these CNVs. It is intended to perform experiments with these agents both immediately after laser treatment to find out whether the formation of CNVs can be prevented and in fully developed CNVs in order to obtain information whether the treatment leads to a reduction of the size of the CNVs. The effect of the VEGF-blocker aflibercept is investigated additionally in an equal manner which serves as an internal control. If the in vitro and in vivo results indicate that certain peptides might be involved in the pathogenesis of these CNVs, the combined treatment of VEGF with particular peptides in vitro and the combined treatment of aflibercept with particular peptide antagonists should be explored in vivo to find out whether the effect is additive. Furthermore, immunoreactivities for the three neuropeptides should be visualized by immunofluorescence in the membranes. Finally, if the peptides are involved in the pathogenesis, the expression of the NK1 receptor and the Y2 receptor including the NPY processing to the Y2 agonist NPY3-36 by endothelial DPPIV should be detected by RT-PCR.

Substance P (SP) and neuropeptide Y (NPY) are neuropeptides which are well kown to act proangiogenic via the neurokinin (NK) -1 and NPY Y2 receptor, respectively. In this project it was explored whether these two receptors are involved in the pathogenesis of laser-induced choroidal neovascularizations (CNVs) in mice. For this purpose, the CNV size was evaluated both by SD-OCT and flat mount preparations in NK1 and Y2 k.o. mice after laser treatment as well as after intravitreal injection of specific nonpeptidergic NK1 and Y2 receptor antagonists both immediately after laser treatment as well as after fully development of CNVs in wildetype mice. The effect of Eylea was equally explored. As a result of this expensive and pretentious animal experiment, the CNV size was found to be significantly reduced in both NK1 and Y2 k.o. mice. Furthermore, intravitreal injection of a Y2 receptor antagonist and to a minor extent also of a NK1 receptor antagonist led to a reduction of the CNV size to a similar extent than Eylea when injected immediately after laser treatment but both antagonists had no effect when injected after fully development of CNVs. In contrast, intravitreal injection of Eylea significantly reduced CNV size not only when injected immediately after laser treatment but also after fully development of CNVs whereas there was no additive effect of combined treatment of Eylea with the peptide receptor antagonists observed. As a conclusion, the NK1 and Y2 receptors mediate the development of laser-induced CNVs in mice to a similar extent as VEGF, most obviously via upregulation of the receptors on choroidal endothelial cells, but they do not play an effective role in the regression of fully developed neovascularizations in this animal model. Consequently, both of these peptidergic systems might be significantly integrated in the initiation of the pathobiological processes in wet age-related macular degeneration.