A New Role for a Krebs Cycle Intermediate : -Ketoglutarate Promotes Retinal Angiogenesis

Abstract

Purpose: : Retinopathy of prematurity (ROP), a major cause of blindness in developed countries, occurs in two phases : the cessation of normal eye development after birth and a subsequent abnormal and exagerated vessel growth. Recently, we presented a previously undisclosed role for the krebs cycle intermediate succinate and its cognate receptor GPR91 in the modulation of retinal vessel. Here we investigated the role of -ketoglutarate and its cognate receptor GPR99 in retinal vessel growth.

Methods: : The retinal expression levels of GPR99 were determined by real-time PCR and western blots. Effects of -ketoglutarate on developmental retinal vascularization were assayed 2 days following intravitreal injection (250µM) of P1, P3 and or P7 Sprague-Dawley rat pups. Areas of central vasobliteration were assessed after intravitreal injection of -ketoglutarate (P5-P7) and the anti-angiogenic effects of SiGPR99 were investigated in the neovascular phase of an oxygen induced model of ROP (80%-10% O₂ : 24hrs cycling protocol). Responsiveness of GPR99 to fluctuating O₂ levels was determined in vitro by real-time PCR and western blot of mixed retinal cell cultures subjected to hypoxia (5%O₂) and angiogenic properties confirmed in aortic ring explants.

Results: : -Ketoglutarate significantly enhanced developmental vascular densities by 15-30% at different time points assayed. Similarly, -ketoglutarate reduced oxygen induced central vasobliteration in the rat model of ROP ; likely by preserving the original vascular bed and alleviating the hypoxic stimulus for growth. These pro-angiogenic properties of -ketoglutarate were confirmed by the sprouting observed in aortic ring explants.

Conclusions: : Our results disclose a novel pro-angiogenic role for -ketoglutarate and its receptor GPR99. These findings provide additional support for the involvement of metabolite signaling in response to the energy compromise observed in ischemic conditions.

Keywords: retinal neovascularization • hypoxia • receptors: pharmacology/physiology