The Role And Mechanism Of Galectin-1 In A Murine Model Of Oxygen Induced Retinopathy

Section one:The distribution,location and expression of galectin-1 in a murine model of oxygen-induced retinopathyObjective:To study the distribution and location of galectin-1(Gal-1),as well as its expression in oxygen-induced retinopathy(OIR)mouse retina via morphological study and molecular biological study.Methods:C57BL/6J mice were randomly divided into the normal control group and OIR group.The OIR mouse model is established according to Smith’s protocol:postnatal day 7(P7)pups were placed under 75%±1%oxygen for 5 days,then back to room air.P17 mice were executed to count the pre-retinal neovascular cells by H&E staining.The RNV,vaso-obliteration(VO)and hypoxic condition of mice in P12,P14,P17,P19 were evaluated by quantification the area of retinal neovascularization tufts,VO area and hypoxic area.The distribution and locatin of Gal-1 was displayed through whole mount retina staining and cryosection staining.Western blot analysis was used to detect the protein level of Gal-1 in both control group and OIR group from P12 to P19.At P17,Western blot and real time PCR was used to compare the protein and mRNA levels of Gal-1 in both groups.Results:The number of pre-retinal neovascular cells in retina from OIR group were obviously higher than those in the control group at P17(P<0.05).The whole mount retina indicated that hypoxic area appeared at P12 and the RNV reached the peak at P17 in OIR retina(P<0.05).Western blot analysis showed that protein level of Gal-1 reached the peak at P17,which is consistent with RNV in OIR.Both western blot and real time PCR showed that Gal-1’s expression are increased significantly in OIR retina at P17 than those in the control group(P<0.05).The distribution of Gal-1 staining indicated that Gal-1 is in a very close relationship with RNV,the cryosection staining showed that Gal-1 located mainly in ganglion cell layer,inner plexiform layer and inner nuclear layer.Conclusion:We successfully established the OIR model.In OIR model,we found that Gal-1’s distribution and location are very close to RNV.The protein level of Gal-1 is consistent with RNV in OIR.These results all indicated that Gal-1 could be a pro-angiogenic agent in RNV.Section two:Suppression of retinal neovascularization by OTX008,a selective inhibitor of Gal-1Objective:To study the inhibory effect on RNV by intravitreal injectin of OTX008,a selective inhibitor of Gal-1.Methods:C57BL/6J mice were randomly divided into four groups:normal control group,OIR-OTX008 group,OIR group and OIR-PBS group.The OIR mouse model is established according to Smith’s protocol:postnatal day 7(P7)pups were exposed to 75%± 1%oxygen for 5 days,then back to room air.In OIR-OTX008 group,mice were intravitreal injected with 1 μl OTX008(0.25 μg/μl)at P12,OIR-PBS group were received the same volume of PBS as a vehicle control.Western blot analysis was used to detect the protein level of Gal-1 four groups.H&E staining and whole-mount retinal staining was used to evaluated the anti-angiogenic effect of OTX008 after inhibition of Gal-1.Results:Intravitreal injection of OTX008 significantly decreased the protein level of Gal-1 than those from OIR group and OIR-PBS group(P<0.05).H&E staining and counting of pre-retinal neovascular cells indicated that OTX008 injectinon significantly reduced the number of RNV compared to those from OIR and OIR-PBS group(P<0.05).Whole-mount retinal staining showed that OTX008 injection obviously inhibited the RNV,vaso-obliteration and hypoxic areas compared to those from OIR and OIR-PBS group(P<0.05).Conclusion:Intravitreal injection of OTX008 could effectively decrease the protein level of Gal-1,in order to inhibit the retinal neovascularization,vaso-obliteration and hypoxic area.All the results indicate that Gal-1 could be a pro-angiogenic agent in OIR.Section three:Construction,packaging and indentification of recombinant adenovirus carrying small interfering RNA of Gal-1Objective:To design and construct a recombined adenovirus carrying siRNA of Gal-1 with enhanced green fluorescent protein tag.Methods:The sequence of Gal-1 RNA interference(RNAi)was designed with OptiRNAi Software,an online system of RNAi templet encoding following the designing principle.These sense and antisense oligonucleotides were ligated to Age I and EcoR I,then annealed to form double strains of DNA that designated Gal-1.The double strains of siRNA targeting Gal-1 was inserted into the linearized plasmid hU6-MCS-Ubi-EGFP that has been already digested with Age I and EcoR I to form the final shuttle plasmid hU6-MCS-Ubi-EGFP.After sequencing,the recombined shuttle plasmid was co-transfected with the genomic plasmid pBHG lox △E1,3Cre into HEK293 cells to obtain the recombined adenovirus Ad-Gal-1-RNAi-EGFP by AdMax system.The recombinant adenovirus was amplified by repeated infection of HEK293 cells and purified by Adeno-X virus purification kit,and the titer determination was done using the end-point dilution method.Results:The recombinant adenovirus Ad-Gal-1-RNAi-EGFP was constructed and confirmed by sequencing.The titer of recombinant adenovirus was 1.0 × 109 PFU/ml.Conclusion:The recombinant adenovirus carrying small interfering RNA of Gal-1 with EGFP tag was constructed successfully,which provided an effective tool to study the role and mechanism of Gal-1 in OIR.Section four:Silencing of Gal-1 inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathyObjective:To investigate the effects on retinal neovascularization of retinal Gal-1 gene silencing through intravitreal injection of Ad-Gal-1-RNAi.Methods:C57BL/6J mice were randomly divided into four groups:normal control group,OIR group,OIR-Gal-1-RNAi group and OIR-GFP group.The OIR mouse model is established according to Smith’s protocol:postnatal day 7(P7)pups were exposed to 75%± 1%oxygen for 5 days,then back to room air.In OIR-Gal-1-RNAi group mice were intravitreal injected with 1 μl of Ad-Gal-1-RNAi at P12,OIR-GFP group were received the same volume of Ad-GFP as a negtive control.At P17,cryosection from normal control group and OIR-Gal-1-RNAi group were stained with GFP antibody to show the transfection of adenovirus.H&E staining was used to detect the pre-retinal neovascular cells.Whole-mount retina influorescence was used to evaluate the alteration of RNV,VO and hypoxic area in four groups.Western blot and real-time PCR was used to assess the protein and mRNA levels of Gal-1.Results:GFP staining showed that adenovirus was successfully tranferred into mouse retina in OIR,especially in ganglion cell layer,inner plexiform layer and inner nuclear layer.Intravitreal injection of Ad-Gal-1-RNAi significantly decreased the protein level of Gal-1 than those from OIR group and OIR-GFP group(P<0.05).H&E staining and counting of pre-retinal neovascular cells indicated that Ad-Gal-1-RNAi injectinon significantly reduced RNV compared to those from OIR and OIR-GFP group(P<0.05).Whole-mount retinal staining showed that Ad-Gal-l-RNAi injection obviously inhibited the RNV,vaso-obliteration and hypoxic area compared to those from OIR and OIR-GFP group(P<0.05).Conclusion:Ad-Gal-1-RNAi transfer via intravitreal injection was efficient in OIR mouse model.Gal-1 silencing in OIR retina significantly reduced RNV and ameliorates retinal hypoxia.Section five:The underlying mechanism of anti-angiogenic effect of Gal-1 silencing in a mouse model of oxygen-induced retinopathyObjective:To investigate the possible mechanism of Gal-1 gene silencing through intravitreal injection of Ad-Gal-1-RNAi on reducing RNV.Methods:C57BL/6J mice were randomly divided into four groups:normal control group,OIR group,OIR-Gal-1-RNAi group and OIR-GFP group.The OIR mouse model is established according to Smith’s protocol:postnatal day 7(P7)pups were exposed to 75%± 1%oxygen for 5 days,then back to room air.In OIR-Gal-1-RNAi group mice were intravitreal injected with 1 μ1 of Ad-Gal-1-RNAi at P12,OIR-GFP group were received the same volume of Ad-GFP as a negtive control.At P17,retinas from four groups were collected to analyze the protein and mRNA level of Nrp-1 and Bcl-2 via Western blot and real-time PCR.Results:The protein level of Nrp-1 and Bcl-2 in OIR-Gal-1-RNAi group was significantly lower than those in OIR group and OIR-GFP group(P<0.05).The mRNA level of Nrp-1 and Bcl-2 in OIR-Gal-1-RNAi group was significantly decreased than those in OIR and OIR-GFP group(P<0.05).Conclusion:Gal-1 gene silencing ameliorates RNV in a mouse model of OIR possibly through reducing the level of its receptor Nrp-1,which down-regulated the expression of Bcl-2.Section six:Vascular normalization by Gal-1 silencing in a murine model of oxygen-induced retinopathyObjective:To investigate the vascular normalization potential of Gal-1 silencing in a murine model of oxygen-induced retinopathy.Methods:C57BL/6J mice were randomly divided into two groups:OIR-Gal-1-RNAi group and OIR-GFP group.The OIR mouse model is established according to Smith’s protocol:postnatal day 7(P7)pups were exposed to 75%± 1%oxygen for 5 days,then back to room air.In OIR-Gal-1-RNAi group mice were intravitreal injected with 1 μl of Ad-Gal-1-RNAi at P12,OIR-GFP group were received the same volume of Ad-GFP as a negtive control.At P17,the vascular normalization was evaluated by whole-mount staining of Concanavalin A,NG2 and CD31.The underlying mechanism of vascular normalization was assessed by the relative phosphorylation of MYPT-1 through western blot analysis.Results:At P17,by increasing retinal vascular perfusion by whole-mount staining of concanavalin A,NG2 and CD31.The western blot analysis indicated that the mechanism of vascular normalization could be through inhibiton of phosphorylation ofMYPT-1.Conclusion:Gal-1 gene silencing ameliorates retinal hypoxia and promote retinal vascular normalization through ROCK pathway inhibiton.