Methallothionein-3 contributes to vascular endothelial growth factor induction in a mouse model of choroidal neovascularization

Abstract

In the present study, we investigated possible roles of the zinc (Zn)-binding protein metallothionein-3 (MT3) and cellular Zn in a mouse model of laser-induced choroidal neovascularization (CNV) using wild-type (WT) and MT3-knockout (KO) mice. Quantitative RT-PCR was used for the detection of MT3 mRNA. CNV was induced in mice between 8 and 12 weeks of age by disrupting the Bruch's membrane using an argon laser. Fundus photography and fluorescein angiography (FA) were performed 2 weeks following laser photocoagulation. The possible connection between MT3 and vascular endothelial growth factor (VEGF) expression was explored by quantifying VEGF levels in WT and MT3-KO mouse retinas by enzyme-linked immunosorbent assay. The role of Zn in VEGF expression was tested in WT and MT3-KO cells treated with pyrithione, with or without additional Zn, using immunoblotting and fluorescence photomicrography. Following laser-treatment, MT3-KO mice exhibited substantially smaller areas of CNV compared to WT mice. In addition, retinal angiograms revealed less severe fluorescein leakage in MT3-KO mice than in WT mice. On day 14 following the induction of CNV, VEGF expression was markedly increased in WT mice, but remained unchanged in MT3-KO mice. Consistent with the possible involvement of Zn released from MT3, raising intracellular Zn levels increased VEGF levels and activated its receptor, Flk-1, in both WT and MT3-KO retinal cells. Present results demonstrated that neural retinal cells express high levels of MT3, which might play a role in the process of CNV development. Moreover, Zn released from MT3 may contribute to VEGF induction.

Physiological Roles of Metallothioneins in Central Nervous System Diseases

“…1). In this study, we confirmed that hypoxia-inducible factor (HIF)-1α was stabilized in the nuclear fraction, but MT-1/2 was increased in the cytosol fraction under hypoxia, and that knockdown of MT-1 or MT-2 increased HIF-1α polyubiquitination 42) (Fig. 1).…”

“…It has been previously indicated that MT-1/2 KO mice decreased expression of the growth factors tumor growth factor-β1 (TGF-β1), NT-3, basic fibroblast growth factor (b-FGF), and VEGF. 53) Furthermore, we first reported the involvement of MT-1/2 in retinal neovascularization 42) (Fig. 1).…”

“…The concentrations of MT-1/2 and VEGF were significantly higher in the vitreous fluid of proliferative diabetic retinopathy (PDR) patients than in that of the macular hole (control group). 42) Clinical data showed the participation in DR pathogenesis of MT-1/2 as well as basic research using mice. Our study is the first report that MT-1/2 could be new molecular targets for retinal neovascularization including DR via HIF-1α degradation.…”

“…Our data suggest that CNV is decreased in MT-1/2 KO mice. 42) On the other hand, MT3, which is expressed at high levels in the neurosensory retina, is involved in both CNV itself and vascular leakage from CNV. 43) MT-1, -2, and -3 might help suppress the pathogenesis and progression of AMD via the mechanism of CNV.…”

Role of Metallothioneins 1 and 2 in Ocular Neovascularization

“…38 However, according to our findings and previous reports, not only MT-1/2-but also MT-3-deficient mice had exacerbated ocular angiogenesis. 39 These findings suggest that MT contributes to decrease of HIF-1a and VEGF expression, and this effect was stronger than the protective effects of RPE through antioxidative stress. Therefore, MT might promote angiogenesis.…”