Retinal Expression Of Wnt-pathway Mediated Genes In Low-density Lipoprotein Receptor-related Protein 5 (Lrp5) Knockout Mice

Abstract

Purpose: : Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5), a Wnt co-receptor, impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. FEVR is a Wnt-related genetic disease in which abnormal retinal vascular development leads to pathological neovascularization and vascular leakage. Similar to human patients, mice lacking Lrp5 (Lrp5^-/-) exhibit delayed and incomplete growth of retinal vessels. In order to gain insight into the molecular mechanisms behind the pathology of FEVR and related eye diseases also impacted by mutations in Wnt signaling, we analyzed gene expression in the developing retinas of Lrp5^-/- mice.

Methods: : Lrp5^-/- and wild type (WT) retinas from postnatal day (P) 8 mice were isolated and either stained with lectin for quantification of vascular growth or processed for RNA isolation (n=6 per group). Retinal and brain samples from both groups were also sectioned and lectin-stained for observance of vasculature. Gene expression levels in total RNA from WT and Lrp5^-/- retinas isolated on P8 were evaluated with an Illumina microarray analysis using the Mouse-WG6 expression BeadChip (n=3 per group), followed by validation with RT-qPCR analysis.

Results: : Retinas from P8 Lrp5^-/- mice exhibited significantly delayed retinal vascular development as compared to WT retinas (WT: 92.2±1.6% vs. Lrp5^-/- : 69.3+2.7%; p≤0.0001). Further, the normal regression of hyaloid vessels and formation of deep layer retinal capillary networks observed in WT mice is absent in the Lrp5^-/- mice. Lrp5^-/- mice also exhibited lower vascular brain density at P8 and developed dilated retinal vasculature with enlarged microaneurysm-like lesions from P12 through adulthood. In the microarray analysis, we found a significant downregulation of tight junction protein claudin5 and amino acid transporter slc38a5 in Lrp5^-/- retina, as well as an increase in expression of endothelial permeability marker plvap. Additionally, we found certain Wnt ligands to be significantly down regulated in Lrp5^-/- retinas, particularly Wnt7b.

Conclusions: : These data suggest that the downregulation of cell adhesion proteins and increase in vessel permeability markers in Lrp5^-/- mice may contribute to their observed defects in retinal vasculature, as well as those found in patients with FEVR. Thus, the Wnt signaling pathway presents as a potential future target for prevention and treatment of vascular eye diseases.

Keywords: candidate gene analysis • retinal development • cell adhesions/cell junctions