ObjectiveTo observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice. MethodsCREB1 siRNA construct was created, screened and packaged to produce CREB1 RNAi-lentivirus. One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group, oxygen induced retinopathy (OIR) group, empty vector group and CREB1 therapy group with 35 mice in each group. Mice in the normal group were kept in normal room air, while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7). The mice in the OIR group were not treated. The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP, 1 μl), and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography. The areas of RNV and non-perfusion region were calculated. The expression of CREB1, phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels, Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. ResultsThe number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P<0.05), while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P<0.05). The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group, while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group. The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220, 110.090; P<0.05). The mRNA expression of CREB1 and protein expression of P-CREB1, the mRNA and protein expression of VEGF-A, Akt, PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group, while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group. The difference of mRNA expression of CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=6.087, 5.464, 6.191, 8.627; P<0.05). The protein expression of P-CREB1, VEGF-A, Akt, PI3K in the retina among 4 groups were significant (F=162.944, 13.861, 19.710, 22.827; P<0.05). ConclusionRNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation.
ObjectiveTo investigate the inhibitory effects of 5-lipoxygenase (5-LOX) on oxygen-induced retinal neovascularization in mice and to explore its possible mechanisms. Methods7-day-old C57BL/6J mice were randomly divided into normal group, oxygen induced retinopathy (OIR) model group, large-dose group, small-dose group and control group with 12 mice in each group. The mice with their mothers were kept in (75±2)% of oxygen environment for 5 days and then returned to normoxia for 5 days to establish the OIR model except for normal group. From postnatal day 12 to 17, the large-dose group and small-dose group received intravitreous injection of 5-LOX at dose of 100 mg/kg and 50 mg/kg respectively, while the control group received the same volume of 1% dimethyl sulfoxide. The mice in the OIR group received no treatment. The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. The mRNA expression of 5-LOX, vascular endothelial growth factor (VEGF)-a, VEGF receptor 2 (VEGFR-2) on retinal tissue were detected by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of 5-LOX, VEGF-a, VEGFR-2 and phosphorylation extracellular signal-regulated kinase (P-ERK) 1/2 on retinal tissue were detected by Western blot. ResultsThe number of vascular cell nuclei breaking through the ILM in the large-dose group and small-dose group decreased significantly compared with the OIR group and control group (F=73.390, P < 0.05). The mRNA expression and protein expression of 5-LOX, VEGFa, VEGFR-2 on retinal tissue were decreased significantly in the large-dose group and small-dose group as compared with the OIR group and control group (F=92.668, P < 0.05). The difference of VEGFR-2 protein expression between large-dose group and small-dose group was not significant (F=2.118, P > 0.05). The differences of 5-LOX, VEGF-a, P-ERK 1/2 protein expression between large-dose group and small-dose group were significant (F=86.490, 165.128, 139.424; P < 0.05). ConclusionHypoxia may induce 5-LOX expression in the retina. Retinal neovascularization was significantly inhibited by selective inhibition of 5-LOX.
ObjectiveTo investigate the inhibitory effects and related mechanism of S100A4 gene silencing on oxygen-induced retinal neovascularization. Methods7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, normal-S100A4 group, oxygen induced retinopathy (OIR) group, OIR-S100A4 group, OIR-green fluorescent protein (GFP) group. To establish the OIR model, mice from all groups except normal one were exposed to (75±2)% oxygen for 5 days and then to room air. In the OIR-S100A4 group and OIR-GFP group, the OIR mice were given an intravitreal injection of 1μl of 1.0×109 PFU/ml adenovirus of Ad-S100A4-RNAi or Ad-GFP at P12, and then returned to normoxia for the next 5 days. In the OIR group, OIR was induced in C57bl/6J mice from P7 to P17. In the normal-S100A4 group, the normal P12 mice were give an intravitreal injection of 1 μl of Ad-S100A4-RNAi adenovirus, and maintained in room air from P12 to P17. In normal group, newborn mouse litters were maintained in room air from P0 to P17 without any treatment. Mice in all five groups were euthanized at P17, and retinas were collected for biochemical assays and morphological study. Retinal neovascularization (RNV) was evaluated by counting the number of pre-retinal neovascular cells and the whole mount immunofluorescent staining of the mouse retina. Protein and mRNA expression levels of S100A4, cAMP responsive element binding protein (CREB), B cell lymphoma-2 (bcl-2), Caspase-3 were determined with western blot and real-time PCR. ResultsThe number of pre-retinal neovascular cell nuclei in retinas from OIR-S100A4 group were obviously lower than those in the retinas from OIR group and OIR-GFP group (t=13.61, 14.64; P < 0.05). In OIR-S100A4 group, the retinal neovascular tufts area and the vaso-oblitertion area were both significantly smaller than those in OIR group and OIR-GFP group (P < 0.05). Protein level of CREB and bcl-2 were significantly down-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05).On the contrary, protein levels of Caspase-3 were up-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05). ConclusionAd-S100A4-RNAi transfer ameliorates RNV in mouse model of OIR maybe through down-regulating the expression of bcl-2 and CREB, and up-regulating the Caspase-3.