Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
Objective To observe the the inhibitory effect of recombined adenovirus mediated delivery of p21 (rAd-p21) on oxygen-induced retinal neovascularization in mice. Methods A total of 56 C57BL/6 mice at the age of seven days were divided into control group, phosphate buffer solution (PBS) group, rAd-p21 group and rAdno purpose gene control (rAd-NC) group, 14 mice in each group. The retinal neovascularization of PBS, rAd-p21and rAd-NC group were induced by oxygen, and received an intravitreal injection 1 mu;l PBS, rAd-p21 and rAd-NC at postnatal day 11, respectively.The rats of control group were not intervened. At postnatal day 17,RNV was determined by retinal flat mounts and retinal section; non-perfusion areas of retina were analyzed by Image-Pro plus 6.0 software; reverse transcription-polymerase chain reaction (RT-PCR) and Western blot was used to measure the mRNA and protein expression of p21 and CDK2. Results Compared with PBS and rAdNC groups, the retinal nonperfusion areas, neovascularization and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in rAd-p21 group were reduced significantly. Nonperfusion areas of retina in rAd-p21 group was less than that in PBS and rAd-NC groups, the difference among these three groups was significantly (F=101.634，P＜0.05). Compared with the other three groups, the level of p21 mRNA and protein in rAd-p21 group increased significantly (F=839.664, 509.817;P＜0.05); the level of CDK2 mRNA and protein in rAd-p21 group decreased significantly (F=301.858, 592.882;P＜0.05). Conclusion rAd-p21can inhibit oxygen-induced retinal neovascularization, up-regulated p21 expression and down-regulated CDK2 expression may be the mechanism.
Objective To investigate the inhibitory effects of 15-lipoxygenase-1 (15-LOX-1) gene transfer on oxygen-induced retinal neovascularization in mice. Methods Ninety-six 7-day-old C57BL/6J mice were randomly divided into normal control group, oxygeninduced retinopathy (OIR) model group, gene treated group and empty vector group. The mice with their mothers were kept in (75plusmn;2) % 02 environment for 5 days and then returned to normoxia for 5 days to establish the OIR model. At postnatal day 12, the gene treated group received intravitreous injection of recombinant adenovirus (Ad) vector containing both enhanced green fluorescent protein (EGFP) and mouse 15-LOX-1 genes (Ad-15-LOX-1-EGFP) at 1 l, while the empty vector group received the same volume of recombinant Ad vector containing EGFP (Ad-EGFP). The expression of EGFP was observed on flat-mounted retina by fluorescence microscopy 2 days after intravitreous injection of Ad-15-LOX-1-EGFP. At postnatal day 17, the efficacy of 15-LOX-1 gene transfer on retinal tissue was detected by immunofluorescence staining, real-time polymerase chain reaction and Western blot. The changes of retinal vessels, relative retinal non-perfusion and neovascularization areas were evaluated by fluorescein isothiocyanate-dextran fluorescein angiography on flatmounted retina. The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. Results Two days after intravitreous injection of Ad-15-LOX-1-EGFP, the expression of EGFP had been seen by fluorescence microscopy on Flat-mounted retina. Immunofluorescence staining of retinal section revealed that 15-LOX-1 expression was primarily in the outer plexiform layer, inner nuclear layer and ganglion cell layer of retina. The 15-LOX-1 protein and mRNA expression levels were higher in gene treated group than those in OIR model group and empty vector group (tprotein=22.74 and 24.13 respectively.tmRNA=12.51 and 13.40 respectively; P＜0.01). The relative retinal non-perfusion and neovascularization areas were significantly smaller in gene treated group than those in OIR model group and empty vector group (tnon-perfusion=16.22 and 14.31 respectively.tneovascularization=9.97 and 9.07 respectively; P＜0.01), and the number of endothelium cell nuclei breaking through the ILM in gene treated group was obviously lower than the other two groups (t=14.25 and 11.62 respectively，P＜0.01). Conclusion 15-LOX-1 gene transfer can decrease the oxygen-induced retinal non-perfusion areas and inhibit the retinal neovascularization in mice.
Objective To investigate the effects of recombinant adeno-associated virus type-2 (rAAV2) mediated delivery of pigment epitheliumderived factor (PEDF) on oxygen-induced retinal neovascularization (OIRNV) in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 mu;l rAAV2-PEDF and rAAV2EGFP into the left eyes (experimental group) and the right eyes (control group). All mice were put into the oxygen box right after the injection to induce the OIRNV model.4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 (P13). Twelve mice underwent retinal angiography with high molecular weight fluoresceindextran, and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative nonperfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software. Results The expression level of PEDF protein was higher in the experimental group than that in the control group.The absolute nonperfusion area was (0.96plusmn;0.22) mm2 in the experimental group and (1.96plusmn;0.34) mm2 in the control group; the difference between the two groups was significant（t=-8.554, P＜0.01）. The relative nonperfusion area was (8.64plusmn;1.52)% in the experimental group and (17.27plusmn;2.98)% in the control group with a significant difference between the two groups (t=-8.97, P＜0.01).The absolute area of retinal neovascularization was (0.37plusmn;0.11) mm2 in the experimental group which was obviously higher than (1.26plusmn;0.38) mm2 in the control group (t=-7.8, P＜0.01); the relative areas in experimental and control groups was (3.96plusmn;0.66)% and (11.45plusmn;2.06)%, respectively, whose difference is apparently(t=-8.51, P＜0.01).The areas of retina neovascularization were (0.11plusmn;0.003) mm2 and (0.41plusmn;0.02) mm2 in the experimental and control groups, respectively, and the difference between the two groups was significant（t=-5.14, P＜0.01）.Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion. rAAV2-PEDF can decrease the retinal non-perfusion areas and inhibit the retinal neovascularization in OIRNV mice.
ObjectiveTo observe the inhibitory effect of intraocular injection of recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor (rAAV-PSF) in oxygen induced retinopathy mice model. MethodsEighteen C57BL/6J mice were divided into 3 groups randomly, including normal group, rAAV-PSF injection group, rAAV injection group. Western blot analysis was applied to detect the transfection expression level of PSF. The other 48 C57BL/6J mice were randomly divided into 4 groups: normal group, ischemia-induced retinopathy (OIR) group, rAAV-PSF treated OIR group, rAAV treated OIR group, 12 mice in each group. Placed all mice (excepted the mice in control group) in cages of (75±2)% oxygen concentration environment for 5 days then moved to a normal environment for 5 days to induced the OIR model. At the 12th day the OIR rAAV-PSF treated OIR group was intravitreal injected with 2 μl 5×1013 pfu/ml rAAV-PSF and the rAAV treated OIR group was treated with intravitreal injection of 2 μl 5×1013 pfu/ml rAAV. The mice in OIR group were left intact after moved out of oxygen cages. Five days after injection, the eyeballs were harvested and retinal sections were stained to count the nuclear of retinal endothelium cells. Western blot analysis was applied to detect the protein level of vascular endothelial growth factor (VEGF) in retina. ResultsThere was a significant difference of the expression PSF between normal group and rAAV-PSF treated group (F=16.05,P=0.001). There was no significant difference of the PSF expression between normal group and rAAV treated group(F=16.05,P=0.890). There was a significant difference of the number of retinal endothelium cells nuclear between normal group and OIR group (F=101.00,P=0.007). There is a significant difference of the number of retinal endothelium cells nuclear between rAAV-PSF treated group and OIR group (F=101.00,P=0v002). There was no significant difference of the number of retinal endothelium cells nuclear between OIR rAAV treated group and OIR group (F=101.00,P=0.550). There was a significant difference of VEGF protein expression between normal group and OIR group (F=13.20,P=0.005), OIR group and rAAV treated OIR group (F=13.20,P=0.001). There was no difference of VEGF protein expression between OIR rAAV treated group and OIR group (F=13.20,P=0.071). ConclusionThe rAAV-PSF vitreous injection can inhibit the expression of VEGF in OIR mice, hence to restrain the proliferation of neovascularization.
Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group,oxygen-induced retinopathy (OIR) group, empty vector group and treated group,14 mices in each group. Except for the control group,the mice in the other groups were exposed to (75plusmn;2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid.The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane（ILM） was observed and counted by optical microscope.Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group.The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups（F=39.006，Plt;0.001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinalneovascularization in OIR.
Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFlt-1 on retinal neovascularization (RNV).Methods Recombinant lentivirus sFlt-1（2-3）and sFlt-1（2-4）expressing the sFlt-1 (2-3) and (2-4) immunoglobulinlike regions of sFlt-1 were constructed. 96 seven-day-old C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1: normal control; group 2: experimental control; group 3: sFlt-1（2-3）; group 4: sFlt-1（2-4）.The mice in group 2-4 were exposed to hyperoxia with （75plusmn;2）% O2 for 5 days and then returned to normoxia with 21% O2;the mice received an intravitreal injection with 1 mu;l virus of empty vector, sFlt-1（2-3）,or sFlt-1（2-4）,respectively. Five days later, all mice underwent perfusion fluorecein angiography and retinal wholemont was made to observe the changes of retinal vessels; retinal sections were stained by hematoxylin and eosin and RNV endothelium cell nucleus were counted; vascular endothelial growth factor(VEGF) and VEGF receptor-2 (KDR/Flk-1) protein were measured by Western blot.Results Seventeen days after birth, the retinal area of fluorescein leakage and RNV, RNV nucleus which breaking through inner limiting membrane in group 3 and 4 were smaller or less than that in group 2（P＜0.01）; while VEGF protein didnprime;t changed much （P＞0.05）the expression of KDR/Flk-1 decreased significantly (P＜0.01). Conclusion sFlt-1（2-3）and sFlt-1（2-4）can inhibit the formation of oxygen-induced RNV,the former virus has a better effect.
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 observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsEighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue.ResultsIn the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20, P＜0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71, P＜0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60; P＜0.05).ConclusionIntravitreal injection of LV-191 inhibits RNV in mice model of OIR possibly through up-regulating p21.