The inhibitory effect of betaine on pathologic retinal neovascularization via suppression of reactive oxygen species mediated VEGF signaling

Abstract

Purpose: Pathologic retinal neovascularization is a leading cause of vision loss at all age groups. Reactive oxygen species (ROS) as well as vascular endothelial growth factor (VEGF) play important roles in pathologic retinal neovascularization. In this study, we tested the hypothesis that betaine inhibits pathologic retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR) via suppression of ROS mediated VEGF signaling.

Methods: OIR was induced in C57BL/6 mice exposing 75% O₂ for 5 days (postnatal day (P) 7-P12). Betaine was intravitreously injected at P14 and PBS was injected in control group (N=7/each group). At P17, neovascular tufts area in OIR retina was analyzed after staining with an anti-isolectin B4 antibody. The toxicity of betaine was evaluated by H&E stain and TUNEL stain in the retina. Cell viability was measured by WST-1 assay in human retinal microvascular endothelilal cells (HRMECs). VEGF-induced wound migration assay and proliferation assay were performed on HRMECs with treatment of betaine. The effect of betaine on VEGF-induced VEGFR-2 auto-phosphorylation and its downstream signaling were evaluated by Western blot analysis for phospho-VEGFR-2, VEGFR-2, phospho-ERK, ERK, phospho-Akt, and Akt. In HREMCs, t-butyl hydroperoxide (tBH) induced ROS production and VEGFA mRNA transcription were evaluated by DCFH-DA and quantitative PCR, respectively.

Results: Intravitreal injection of betaine effectively reduced the area of neovascular tufts in OIR retina (68.0±6.7% of the control eye, P=0.027). Even in a high concentration, betaine never induced any structural changes or toxicity in the retina or cytotoxicity in HRMECs. In vitro, betaine significantly inhibited both VEGF-induced migration and proliferation in HRMECs. In addition, betaine suppressed VEGF-induced ERK phosphorylation in HRMECs. Interestingly, betaine reduced tBH-induced ROS production. Moreover, it also attenuated tBH-induced VEGFA mRNA transcription via suppression of ROS.

Conclusions: These data suggest that betaine has an anti-angiogenic effect on pathologic retinal neovascularization via suppression of ROS mediated VEGF signaling. Betaine could be a potent anti-angiogenic agent to treat pathologic retinal neovascularization.