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Steven E. Brooks, Wenbo Zhang, Hua Liu, Zhimin Xu, Harumasa Yokota, Robert W. Caldwell, Akitoshi Yoshida, Ruth B. Caldwell; Hyperoxia Causes Regression of Established Vitreous Neovascularization By Targeting the VEGF/VEGFR-2 Survival Pathway. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2531.
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We have previously shown that hyperoxia treatment (HT) can prevent vitreous neovascularization (NV) while simultaneously allowing retinal revascularization. Here we show that HT can selectively target established vitreous NV in a mouse model of ischemic retinopathy by blocking the VEGF/VEGFR-2, but not the VEGF/VEGFR-1, survival pathway.
P17 mice demonstrating vitreous NV in an established mouse model of ischemic retinopathy were treated with 75% oxygen (HT), VEGF trap, or selective VEGFR-2 blockers (VEGFR-2 inhibitor or VEGFR-2 siRNA). Intra-retinal and vitreous NV were assessed in lectin stained retinal whole mounts using confocal microscopy and image analysis software. Apoptosis was measured by immuno-labeling for cleaved caspase-3. The rescue effects of intravitreal VEGF-A, VEGF-E (specific for VEGFR-2), or PlGF-1 (specific for VEGFR-1) were evaluated in mice receiving HT. VEGFR-2 expression in NV tufts and vessels was assessed in by quantitative RT-PCR and immuno-labeling. Macrophages/microglia were identified by Iba-1 immuno-labeling.
HT reduced vitreous NV by 70% (p<0.0001) within 24 hrs, while increasing avascular area by only 21% (p<0.05). Robust expression of cleaved caspase-3 was noted in NV by 12hrs, and was associated with macrophage/microglial infiltration into NV tufts. Intra-vitreal injection of VEGF-A (p<0.02) or VEGF-E (p<0.01), but not PlGF-1, prevented the HT-induced apoptosis. VEGFR-2 expression in vitreous NV was markedly reduced by HT (p<0.01), but was significantly increased in non-vascular cells (p<0.05). VEGF trap and VEGFR-2 blockers administered on P17 were also effective in causing NV regression (p<0.05), though less effectively than HT.
HT selectively targets pathological NV by inhibiting VEGFR-2 activity and expression, causing rapid onset of apoptosis and macrophage/microglial infiltration. The relatively higher sensitivity to HT of vitreous NV compared to intraretinal NV may be due to its increased dependence on VEGFR-2 activity for survival, a characteristic suggested by the relatively high level of VEGFR-2 expression in pathological NV. This feature may lead to the discovery of selective anti-angiogenic therapies, including HT, which specifically target vitreous NV, while permitting or even enhancing physiologic vascular repair in ischemic retinopathy.
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