Anti-Angiogenic Effect of Metformin in Mouse Oxygen-Induced Retinopathy Is Mediated by Reducing Levels of the Vascular Endothelial Growth Factor Receptor Flk-1

PURPOSE: To evaluate the effect of metformin on vascular changes in oxygen-induced retinopathy (OIR) in mouse, and to elucidate the possible underlying mechanism. METHODS: OIR mice were treated with metformin by intraperitoneal injection from postnatal day 12 (P12) to P17 or P21. At P17 and P21, vessel formation and avascular areas were assessed using retinal flat mounts. Levels of vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assays, and the effects of metformin on VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) were assessed. The effects of metformin on the levels of Flk1 (VEGF receptor-2) and phosphorylated Flk1 (pFlk1) were measured by Western blotting (HUVECs) and immunohistochemistry (retinal tissue). RESULTS: Retinal morphologic changes were analyzed between two groups (saline-treated OIR; metformin-treated OIR). Metformin treatment did not change the extent of avascular areas at P17. However, at P21, when OIR pathology was markedly improved in the saline-treated group, OIR pathology still remained in the metformin-treated OIR group. VEGF expression levels did not differ between metformin- and saline-treated OIR groups at P17 and P21, but Flk1 levels were significantly reduced in the metformin group compared with saline-treated OIR group. Moreover, metformin inhibited VEGF-induced cell proliferation and decreased levels of Flk1 and pFlk1, consistent with the interpretation that metformin inhibits vascular growth by reducing Flk1 levels. CONCLUSION: Metformin exerts anti-angiogenesis effects and delays the normal vessel formation in the recovery phase of OIR in mice, likely by suppressing the levels of Flk1.