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Objective— The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor–like growth factor) plays in ocular angiogenesis. Approach and Results— We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF–induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. Conclusions— These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration.

Hypoxic stress is a risk factor of ocular neovascularization. Hypoxia visualization may provide clues regarding the underlying cause of angiogenesis. Recently, we developed a hypoxia-specific probe, protein transduction domain-oxygen-dependent degradation domain-HaloTag-Rhodamine (POH-Rhodamine). In this study, we observed the localization of HIF-1α proteins by immunohistochemistry and the fluorescence of POH-Rhodamine on RPE-choroid flat mounts. Moreover, we compared the localization of POH-Rhodamine with pimonidazole which is a standard reagent for detecting hypoxia. Next, we investigated the effects of triamcinolone acetonide (TAAC) against visual function that was evaluated by recording electroretinogram (ERG) and choroidal neovascularization (CNV) development. Mice were given laser-induced CNV using a diode laser and treated with intravitreal injection of TAAC. Finally, we investigated POH-Rhodamine on CNV treated with TAAC. In this study, the fluorescence of POH-Rhodamine and HIF-1α were co-localized in laser-irradiated sites and both the POH-Rhodamine and pimonidazole fluorescent areas were almost the same. Intravitreal injection of TAAC restored the reduced ERG b-wave but not the a-wave and decreased the mean CNV area. Furthermore, the area of the POH-Rhodamine-positive cells decreased. These findings indicate that POH-Rhodamine is useful for evaluating tissue hypoxia in a laser-induced CNV model, suggesting that TAAC suppressed CNV through tissue hypoxia improvement.

PURPOSE The incidence of blindness is increasing, in part, because of abnormal ocular neovascularization. Anti-VEGF therapies have yielded impressive results; however, they are not a cure for blindness. Recently, metallothioneins (MTs) 1 and 2 have been implicated in the process of angiogenesis. Therefore, we investigated whether MT-1 and MT-2 were also involved in ocular neovascularization. METHODS The concentrations of MT-1 and MT-2 (hereafter MT-1/2) were observed by ELISA. We examined the role of MT-1/2 in ocular neovascularization by using both an oxygen-induced retinopathy (OIR) model and a laser-induced choroidal neovascularization (CNV) model. We investigated the localization of MT-1/2 in retina. Furthermore, we investigated the expression of hypoxia-inducible factor (HIF)-1α and VEGF in OIR. In vitro, we investigated the degradation of HIF-1α. RESULTS The MT-1/2 were significantly elevated in proliferative diabetic retinopathy patients. Ocular neovascularization, which was induced in both the OIR model and the CNV model, was decreased in MT-1/2 knockout (KO) mice. We confirmed that although MT-1/2 was expressed throughout the murine retina, its expression levels were highest in the endothelial cells. Further, OIR enhanced MT-1/2 expression in the retina. Interestingly, in the OIR model, both HIF-1α and VEGF levels were significantly decreased in the retina of MT-1/2 KO mice. In addition, we found that knockdown of MT-1/2 accelerated ubiquitination of HIF-1α. CONCLUSIONS These results indicate that MT-1/2 are involved in retinal and choroidal neovascularization, and that MT-1/2 might be a new therapeutic target in diseases in which ocular angiogenesis is implicated.