Retinas Of Oxygen-induced Retinopathy Mice Research Articles

Heparanase and Vascular Endothelial Growth Factor Expression Is Increased in Hypoxia-Induced Retinal Neovascularization

Heparanase and VEGF are related closely to angiogenesis in cancer. The purpose of our study was to evaluate the expression and correlation of heparanase and VEGF in hypoxia-induced retinal neovascularization. C57BL/6 oxygen-induced retinopathy (OIR) mice and human retinal microvascular endothelial cells (HRECs) were treated with the hypoxia mimetic agent cobalt chloride (CoCl₂), and in the presence of the heparanase inhibitor phosphomannopentaose sulfate (Muparfostat, PI-88). Heparanase activity was assayed in HRECs, and the expression of heparanase, VEGF protein and mRNA were evaluated by immunofluorescence, ELISA, Western blot, and real-time PCR while retinal flat mounts were used to evaluate the area of neovascularization of mice retina. HREC heparanase activity was increased by treatment with CoCl₂, but was decreased by PI-88. Immunofluorescence showed that heparanase and VEGF staining was intense in hypoxia-treated HRECs and OIR mice retina, while VEGF staining was faint in the normoxia and PI-88-treated ones. Western blot and real-time PCR results indicated that the expression of heparanase and VEGF was increased under hypoxic conditions, and the increase of VEGF was inhibited by PI-88. Retinal flat mounts showed that the area of new vessels in retina of OIR mice was increased compared to the normoxic mice, and this effect was inhibited by PI-88. Heparanase is upregulated and associated with the VEGF expression in hypoxia-induced retinal diseases. Heparanase is involved in hypoxia-induced neovascularization through promoting VEGF expression and may be a new therapeutic target for hypoxia-induced neovascularization retinal diseases.

Ephrin-A4 Is Involved in Retinal Neovascularization by Regulating the VEGF Signaling Pathway

Retinal neovascularization (NV) is a major cause of blindness. Recent research suggests that factors other than VEGF participate in this process. This study aimed to determine the role of ephrin-A4 in retinal NV. The expression and effect of ephrin-A4 was investigated in a mouse model of oxygen-induced retinopathy (OIR) and the RF/6A retina endothelial cell line. Ephrin-A4 expression and VEGF signaling pathway phosphorylation were determined by PCR, immunohistochemistry, and western blot analyses. ShRNA was used to silence ephrin-A4 in vitro and in vivo. Retinal flat mounts and tube formation assays were performed to evaluate ephrin-A4 function in the NV process in vivo and in vitro. Ephrin-A4 was overexpressed in the retina of OIR mice and in RF/6A and RPE cells after CoCl₂ stimulation. In vitro, Ephrin-A4/Fc treatment significantly increased the tube number of RF/6A cells on a membrane preparation and the phosphorylation levels of VEGR2, Akt1, and ERK1/2 in RF/6A cells. Moreover, ephrin-A4 knockout markedly suppressed pathologic neovascularization in vivo and inhibited the proliferation and tube formation capacity of RF/6A cells in vitro. Furthermore, in the absence of ephrin-A4, the phosphorylation of VEGFR2, Akt1, and ERK1/2 was defective under VEGF₁₆₅ stimulation, and the proangiogenic function of VEGF₁₆₅ was also compromised. This study suggests that ephrin-A4 plays an important role in retinal NV and is a potential target against retinal NV. The proangiogenic function of ephrin-A4 may be linked to its crucial role in the VEGF signaling pathway.

Suppression of Retinal Neovascularization by Lentivirus-Mediated Netrin-1 Small Hairpin RNA

Objectives: The function of netrin-1 in pathological angiogenesis and its role in retinal neovascularization were investigated in the retinas of oxygen-induced retinopathy (OIR) mice by inhibition of netrin-1. Methods: Expression of netrin-1 mRNA and protein in the retinas of OIR mice was analyzed by quantitative RT-PCR and immunoblotting. Inhibition of retinal neovascularization was achieved by lentivirus-mediated netrin-1 small hairpin RNA (shRNA) infection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections. Results: Both mRNA and protein expression of netrin-1 were significantly upregulated in postnatal day 17 OIR mouse retinas. Treatment of OIR mice with specific lentivirus-mediated netrin-1 shRNA dramatically reduced neovascular outgrowth into the inner limiting membrane. Neovascular tufts and nonperfused areas were also reduced. Conclusions: High expression of netrin-1 was detected in the retina under ischemic conditions and played a significant role in pathological retinal angiogenesis. Therefore, netrin-1 represents a potential therapeutic target for diabetic retinopathy, retinopathy of prematurity and other ocular neovascular diseases.

Role of unc5b in retinal neovascularization in mice with oxygen-induced retinopathy.

To explore the role of unc5b in retinal neovascularization in murine oxygen-induced retinopathy (OIR). On postnatal 7(P7), C57BL/6J mice were exposed to 75%±2% oxygen for 5 days. On postnatal 12(P12), the mice were brought back to the room air (21% oxygen) to induce retinal neovascularization. Western blot analysis was performed to examine the temporal expression of unc5b in murine retinas. Double staining for unc5b and isolectin B4 were employed to determine the location of unc5b in murine retinas. The effect of unc5b on retinal neovascularization was evaluated by intravitreal injection of unc5b-FC in mice with OIR. Retinal neovascularization was measured by counting neovascular cell nuclei above the internal limiting membrane and by angiography of flat-mounted retinas perfused with fluorescein dextran. Compared to age-matched normal mice, the expression of unc5b was significantly increased in retinas of OIR mice on P17 and P21. Unc5b was apparently expressed in retinal vessels of OIR while being negative in normal retinal vessels. Retinal neovascularization in eyes injected with unc5b-FC was significantly reduced. Unc5b-FC can effectively inhibit retinal neovascularization induced by OIR. It may serve as a powerful and novel therapy for ischemia-induced retinal disease.