An assessment of the angiogenic effects of VEGF-A and ANGPTL4 in the mouse retina.

Abstract

Purpose : To investigate the effects of VEGF-A and ANGPTL4 on retinal angiogenesis and blood vessel leak.

Methods : Adult (>8 week old) C57Bl/6 mice were injected intravitreally with VEGF-A or ANGPTL4 and the effects were monitored 24, 48 and 72 hours post-injection using FA and OCT (IFO). In the Oxygen-Induced Retinopathy (OIR) model, neonatal (P6) C57Bl/6 pups maintained at high oxygen environment (75% O2) for 5 days till P11 were injected systemically with 25 mg/kg of control hFc protein or ANGPTL4 at P12. Eyes from these mice were harvested at P16, retinas were dissected, stained using GS Lectin-I, imaged and analyzed. In another set of experiments, the effects of ANGPTL4 on Human Umbilical Vein Endothelial Cell (HUVEC) sprouting were assessed in the fibrin bead assay.

Results : Intravitreal injections of VEGF-A rapidly produced significant leak that persisted for at least 48 hours while intravitreal injections of ANGPTL4 had no effect on blood vessel leak. To test if the described pro-angiogenic effect of ANGPTL4 requires pre-activation of the endothelium with VEGF-A, the two proteins were injected sequentially. First, VEGF-A was injected alone and 48 hours later, ANGPTL4 was injected into the same eye. However even in these experiments ANGPTL4 did not show any additional pro-angiogenic effect.

In the OIR model, ANGPTL4 did not show any effect on the normal and pathological angiogenesis.

In the fibrin bead assay model, VEGF produced a robust cell sprouting response, while ANGPTL4 did not induce endothelial cell sprouting. Additionally, ANGPTL4 did not inhibit VEGF-induced endothelial cell sprouting, while the VEGF blocker aflibercept quantitatively suppressed the endothelial cell sprouting response.

Conclusions : VEGF-A injection induced retinal blood leak 24 hours post-injection, which started to decrease by 48 hours and disappeared almost completely by 72 hours. VEGF-A also induced endothelial cell sprouting. Despite the published data, no angiogenic effect of ANGPTL4 was observed in all these experiments. The procedural differences that may explain the distinction between our results and contrary ones in the literature remain to be elucidated.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.