Flt-1 Activation Prevents Oxygen-induced Vascular Loss in ROP without Promoting Vaso-proliferation

Abstract

Abstract: : Purpose: To determine the roles of Flt-1 and Flk-1 in retinal vascular survival and proliferation in the mouse retinopathy model. In neonates, retinal vessels are susceptible to oxygen-induced apoptosis, initiating retinopathy of prematurity. Exogenous VEGF, which binds to Flt-1, Flk-1 and neuropilin, can prevent oxygen-induced loss as well as stimulate vaso-proliferation. The receptors involved in survival have not been well defined. It would be of great clinical importance to find an agent to prevent retinal vessel loss without stimulating vessel proliferation. Such an intervention might safely prevent retinopathy of prematurity (ROP) and perhaps diabetic retinopathy. Methods: Real time RT-PCR was used to quantify mRNA of Flt-1, Flk-1, PlGF-1, and VEGF in C57 Bl6 mouse retina during development from P0 to P26. Retinal whole mount in situ hybridization of Flt-1 and Flk-1 and VEGF was used to localize mRNA expression. Intravitreal injections of Flt-1 specific ligand PlGF-1 and Flk-1 specific ligand VEGF-E were used to evaluate the role of Flt-1 and Flk-1 in oxygen-induced vaso-obliteration, revascularization, and neovascularization in the mouse retinopathy model. Results: At P5 in mouse retina, Flt-1 protein coincides with retinal vessels, whereas Flk-1 is seen only in neural retina. Real-time RT-PCR showed a 60-fold induction of Flt-1 during vessel development (P3 vs. P26) in mouse retinas. The Flk-1 expression level was almost 200 times higher than Flt-1 in avascular P3 mice retina and the expression level was unchanged throughout the retinal vessel development. Intra-vitreal injections of the Flt-1 specific ligand, human PlGF-1, prior to the onset of 17 h hyperoxia stress decreased hyperoxia-induced vaso-obliteration from 22.2% to 5.1%, whereas injections of the Flk-1 specific ligand VEGF-E showed no effect. Furthermore, PlGF-1 stimulation of Flt-1 did not induce endothelial cell proliferation during normal vessel growth, during revascularization nor during hypoxia-induced vaso-proliferation. Conclusions: Activation of Flt-1 but not Flk-1 protects against oxygen-induced vessel loss. Flt-1 activation does not stimulate retinal vascular proliferation and neovascularization in vivo. Thus, this study differentiates the role of Flt-1 and Flk-1 in oxygen-induced vessel loss and provides evidence for a survival role of Flt-1 in retinal vessel degeneration, and reveals Flt-1 as a potential new target for control of vaso-obliteration in ROP and perhaps other retinopathies

Keywords: apoptosis/cell death • retinopathy of prematurity • diabetic retinopathy