Role of the Krebs Cycle Metabolites in Retinal Angiogenesis

Abstract

PurposeRetinopathy 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 exaggerated vessel growth. Peripheral hypoxia occur after the first phase in premature eye and induce production of many growth factors implicated in this second destructive neovascularization phase. Our laboratory has previously demonstrated the role of succinate/GPR91 in the modulation of retinal vessel growth. Here, we investigated the role of α‐ketoglutarate (α‐KG) and its cognate receptor GPR99 in retinal angiogenesis.MethodsEffects of α‐KG on developmental retinal vascularization were assessed following intravitreal injection of Sprague‐Dawley rat pups in development. Moreover, the effects of a siGPR99 knockdown following intravitreal injection were investigated in the neovascular phase of ROP. Endogenous expression of retinal GPR99 was examined in Sprague‐Dawley rat retinas by immunohistochemistry. In vitro, we investigated the expression of GPR99 and pro‐angiogenic factors by Western blot and PCR analysis. The ability of α‐KG to promote vessel sprouting was determined by aortic rings cultured.Resultsα‐KG significantly enhanced developmental vascular densities at different time points. Neovascularization was blocked by a siGPR99 and allowed normal vessel growth in ROP. Moreover, GPR99 was robustly expressed in RGCs as confirmed by co‐labeling with neuron markers and, in vitro, mainly in neuronal cells. Conditioned media from α‐KG stimulated‐neurons induced aortic rings sprouting, in correlation with an up‐regulation of pro‐angiogenic factors expression.ConclusionsOur results disclose a pro‐angiogenic role for α‐KG and its receptor GPR99, providing additional supports for the involvement of metabolite signalling in ischemic conditions.