Eri Suganami

Transcription Factor Ets-1 Mediates Ischemia- and Vascular Endothelial Growth Factor-Dependent Retinal Neovascularization

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Role of vitronectin receptor-type integrins and osteopontin in ischemia-induced retinal neovascularization.

PURPOSE It has been reported that vitronectin receptor-type integrins mediate vascular cell proliferation and migration. In this study, we investigated the expression of vitronectin receptor-type integrins and osteopontin in ischemia-induced retinal neovascularization, and examined the role of osteopontin in angiogenesis as a ligand of vitronectin receptor-type integrins. METHODS Retinal neovascularization was produced by exposing C57BL/6J mice to 75% oxygen from postnatal day (P) 7 to P12. Expression of vitronectin receptor-type integrins and osteopontin was assessed by Northern blot analysis, in situ hybridization, and immunofluorescence. The role of osteopontin in retinal angiogenesis was evaluated by tube formation assay using cultured bovine retinal microcapillary endothelial cells. RESULTS In the murine model, integrin alpha(v) mRNA was increased from P14 with a 2.6-fold peak response observed on P19, when retinal neovascularization was remarkable. Indirect immunofluorescence for vitronectin receptor-type integrins revealed prominent expression of integrin alpha(v)beta3/beta5 in the neovascular endothelial cells. Osteopontin mRNA was increased from P14, with a 2.0-fold peak response observed on P19. In situ hybridization demonstrated localization of osteopontin mRNA in neovascular tufts. Vascular endothelial growth factor-induced tube formation (8.3 +/- 0.6 mm/field) was inhibited significantly by treatment with anti-osteopontin antibody (4.8 +/- 0.7 mm/field, P <.001). CONCLUSIONS These data suggest that increased expression of both vitronectin receptor-type integrins and osteopontin in ischemic retina contribute to vascular endothelial cell proliferation and to retinal vascular formation by promoting interaction between endothelial cells and extracellular matrix, which leads to retinal neovascularization.