Succinate and -Ketoglutarate Act as Sensors of Hypoxia and Mediate Retinal Angiogenesis

Abstract

Purpose:: The accumulation of the Krebs cycle intermediates succinate and α-ketoglutarate is observed in conditions linked with the deterioration of blood supply, such as ischemia. Succinate was recently identified as the ligand for the orphan receptor GPR91, and α-ketoglutarate for the orphan receptor GPR99. Although Krebs cycle intermediates are known to accumulate in hypoxia/ischemia, their role in retinal angiogenesis remains unknown. We wanted to investigate whether succinate and α-ketoglutarate act as sensors for ischemia and are implicated in restoring blood flow in the injured tissue.

Methods:: Succinate or α-ketoglutarate-induced cell proliferation was assessed by a [H³]Thymidine incorporation assay. Ex vivo experiments were also performed by treating rat aortic rings with the Krebs cycle intermediates. Rat pups were intravitreally injected with succinate and/or α-ketoglutarate at postnatal day 1 (P1) and retinal vascular density was quantified at P3 by lectin staining (endothelial cell-specific marker). The role of GPR91 and GPR99 in angiogenesis was investigated by treating cells, aortic rings or retinas with siRNA against either receptor.

Results:: In vitro, succinate and/or α-ketoglutarate induced a considerable upregulation in the proliferation of human umbilical vein endothelial cells (HUVEC). Consistently, ex vivo experiments performed on aortic rings demonstrated enhanced vessel sprouting following stimulation with succinate and/or α-ketoglutarate, reinforcing their possible involvement in the angiogenesis process. In addition, succinate and/or α-ketoglutarate were injected intraocularly in neonatal pups and exhibited a significant induction in retinal angiogenesis. In RNA interference knockdown experiments, GPR91 and GPR99 were necessary for the proliferation of HUVECs, the vascular sprouting of aortic rings, and the vascularization of neonatal retina.

Conclusions:: Taken together, these findings support significant roles for succinate and α-ketoglutarate and their respective receptors in mediating angiogenesis, and suggest that the inhibition of these GPCRs may be important therapeutic targets in treating ischemic diseases.

Keywords: retinal development • hypoxia • vascular cells