Role of netrin-4 in a Mouse Model of Choroidal Neovascularization

Abstract

Purpose: Netrins are a laminin-like family of proteins that first were characterized as axon guidance molecules in neurogenesis. Interestingly, current research now draws parallels between molecular mechanisms of neurogenesis and angiogenesis claiming that both processes share molecular mechanisms. In this work, we aimed to demonstrate whether netrin-4, the newest member of the netrin-family, joins in the complex mechanisms of choroidal neovascularization (CNV) as part of the pathogenesis of wet Age-related macular degeneration (AMD).

Methods: Exploiting an established laser-induced mouse model of CNV in Ntn-4^-/- mice and C57BL/6J mice as controls, we examined eyes 14 days after laser exposure by fluorescein angiography in order to evaluate the leakage of nascent pathological choroidal blood vessels. Whole flatmounts of the choroid were dissected and stained with an endothelial cell staining (Isolectin IB4) and CD11b as a myeloid cell marker. The size of the laser induced CNV area was measured, the CD11b positive cells counted and statistically analyzed. To prove that the Ntn-4^-/- mice lack netrin-4 expression, immunohistochemical stainings on cross sections on both groups were performed in combination with an immunohistochemical staining of endothelial cells.

Results: Quantification of the fluorescein leakage from choroidal laser scars as a mete of pathological blood vessel formation showed no significant differences between control and Ntn-4^-/- mice. Two weeks after laser coagulation the mean size of the CNV areas of 35824.33 ± 38428.49 μm in control mice (n = 26) did not differ from that in Ntn-4^-/- mice (n = 31) of 24209.96 ± 15199.35 μm (p = 0.064). Also the number of CD11b positive cells in CNV lesions in both groups revealed no significant statistical difference (p = 0.45). Likewise, there was no significant difference in VEGF expression in the sclera/choroid or in the retina of laser-treated Ntn-4^-/- mice when compared to the control mice.

Conclusions: Our data show that the presence of netrin-4 does not affect the formation of pathological blood vessels in this laser-induced mouse model of the wet form of AMD. We hypothesize that netrin-4 does not play a significant role in the pathogenesis of the wet form of AMD.

Keywords: 412 age-related macular degeneration • 453 choroid: neovascularization • 578 laser