Inhibition of Retinal Vascular Development and Vascular Leakage in Pdgfb-iCreERT2Flk1loxP Mice

Abstract

Purpose: : The superficial retinal vascular plexus of mice is formed during the first week after birth. Flk1 is the main signaling receptor for vascular endothelial growth factor during embryonic development and adult neovascularization. To investigate the role of Flk1 in the retinal vasculature, we generated Pdgfb-iCreER^T2Flk1^loxPmice and analyzed vessel growth and leakiness at post-gestation day 6.5 (P6.5).

Methods: : Flk1^loxP mice were crossed with Pdgfb-iCreER^T2 mice to establish Pdgfb-iCreER^T2Flk1^loxP mice. Expression of the Pdgfb-iCreER^T2 transgene is specific to vascular endothelial cells. Tamoxifen was injected intraperitoneally to induce nuclear translocation of the Pdgfb-iCreER^T2 and excision of Flk-1. The mice were sacrificed at P6.5 and the eyes were enucleated and fixed in 4% paraformaldehyde (PFA). The retina was dissected and flat-mounted. To assess the permeability of retinal vessels, mice were perfused with 10 µL/g body weight of 70-kDa lysine-conjugated FITC-dextran (5.0 mg/mL). After 30 seconds, the left ventricle was cut to stop the circulation and the eyes were placed in 4% PFA. Vascular development was evaluated by analyzing the percentage of vascularized retina relative to total retina, and by counting the number of capillaries in specific zones. The permeability was evaluated by quantifying FITC-dextran leakage.

Results: : In Pdgfb-iCreER^T2Flk1^loxP/+ mice, vascular spreading and capillary number were reduced relative to WT mice. In Pdgfb-iCreER^T2Flk1^{loxP/ loxP} mice, there was severely diminished vascular development including no filopodia at the leading edge of tip cells. FITC-dextran leakage was restricted to the distal zone of stalk cells at the leading edge of capillary in WT mice and Pdgfb-iCreER^T2Flk1^loxP/+ mice, but the vessels of Pdgfb-CreER^T2Flk-1^{loxP/ loxP} mice were filled with FITC-dextran suggesting diminished vascular leakage.

Conclusions: : Endothelial Flk1 is essential for development of retinal vasculature. Furthermore, the leakage of FITC-dextran in WT animals could be used to mark the zone of stalk cells at the leading edge of capillaries in the process of retinal vascular development.

Keywords: retinal development • vascular endothelial growth factor