The Research Of Anti-angiogenesis Of Vascular Endothelial Growth Inhibitor In Diabetic Retinopathy

ObjectiveDiabetic Retinopathy(DR)is one of the most common microvascular complications of diabetes and has become a major blindness eye disease.Angiogenesis is the key to DR.The angiogenesis process is controlled by changing the balance between the vascular growth factor and the vascular inhibitory factor.Therefore,looking for an ideal angiogenesis inhibitor and regulating the balance between the vascular growth factor and the inhibitory factor have become the key to the prevention and treatment of DR.VEGI is a new angiogenic antagonist,which can inhibit the proliferation of endothelial cells,angiogenesis and tumor growth.Studies have shown that VEGI can promote the degradation of mFlt1 and up-regulate the expression of sFlt1,thus inhibiting the VEGF and the negative regulation of angiogenesis.This study will explore the effect of VEGI on retinal vascular endothelial cells,which will provide a new direction for the treatment of DR.This subject first explores the difference in expression of VEGI in the vitreous body of patients with PDR,NPDR and non DM.Then,bovine retinal vascular endothelial cells were used to study overexpression of VEGI in high glucose state,and to detect cell proliferation,migration,angiogenesis and cytokines.Construction of a model of retinal neovascularization in mice.We will give mice the subretinal injection of VEGI siRNA and VEGI Lentivirus Expression Vector.The inhibitory effect of OCT and IVIS on retinal vascular leakage and angiogenesis.This study will provide a theoretical basis for the improvement of DR lesions in VEGI.MethodsThis topic is divided into four parts.Part 1 :Differential expression of VEGI in the vitreous body of patients with proliferative diabetic retinopathy and non-proliferative diabetic retinopathy16 cases of non DM patients,19 cases of NPDR in DM and 12 cases of PDR patients who have been treated with surgical treatment from the patients of vitreo-retina surgery in Second Hospital Affiliated to Tianjin Medical University and Tianjin Eye hospital.The vitreous tissue of the patient is left in the dry ice during the operation.The subjects signed the informed consent notice before the experiment to prove the purpose of this study,and to know the necessary research process,and would like to join this study.At the same time,the patient's blood sugar,blood lipid,liver and kidney function and other clinical indicators.The Western blot detection technique was used to detect the expression of VEGF and VEGI among three groups.Part 2 Expression of VEGI in diabetic retinopathy of miceBecause we are unable to obtain human retinal specimens,we can not detect the changes in the expression of VEGF and VEGI in the retina of diabetic patients.Therefore,we use STZ induced diabetic mice as a model of type 1 DM mice,and high fat fed KK-Ay mice as a model of type 2 DM mice.OCT was used to detect the condition of live retinopathy in mice,and HE and WB were used to detect the expression of VEGI and VEGF,as well as the expression of VEGFR1 and VEGFR2.Part 3 Effects of VEGI on the proliferation,migration,transformation of vascular endothelial cells and the corresponding cytokines in retinal vascular endothelial cellsAfter overexpression of VEGI in high glucose state,cell proliferation,migration,tubular changes,and changes of the corresponding cytokines were detected.Cultured human retinal microvascular endothelial cells treated with different concentrations of glucose(25mM,30 mM,50mM),transfection of pcDNA3.1+/VEGI/GFP plasmid and pcDNA3.1 /GFP plasmid after application of CCK8,migration,mitragel,etc.were used to detect cell proliferation,migration and tube formation changes.Part 4 Study on the effect of VEGI on hyperoxia induced retinal neovascularization in miceThe model of retinal neovascularization in mice was constructed.Newborn C57BL/6J mice were randomly divided into normal group,model group and VEGI intervention group.Model group and VEGI intervention group were fed under high oxygen environment.Retinal vascular changes were observed by HE staining,fluorescein perfusion and retinal paving.Results1.In our randomly selected clinical specimens,there was no significant difference in clinical index between DM group and control group.Glycosylated hemoglobin(HbA1c)in patients with DM was significantly higher than that in control group(P< 0.01).Compared with group T2DM+PDR,the level of HbA1 c in group T2DM+PDR was higher than that in group T2DM+NPDR(P< 0.05).2.Compared with non-diabetic group,the expression level of VEGI protein in vitreous body of group DM was significantly reduced.In group T2DM+NPDR,compared with group T2DM+PDR,the level of VEGI in vitreous group of T2DM+PDR group was further reduced.Compared with non-diabetic group and T2DM+NPDR group,the protein expression level of VEGF in vitreous body of T2DM+PDR group increased significantly.Compared with non-diabetic group,the protein expression level of VEGF in T2DM+NPDR group increased significantly.3.Compared with non-diabetic group,the ratio of VEGI/VEGF in vitreous body of group DM was significantly reduced.In group T2DM+NPDR,compared with group T2DM+PDR,the ratio of VEGI/VEGF in vitreous group of T2DM+PDR group decreased more obviously.4.Compared with the control group,the blood glucose of STZ rats and KK-Ay mice increased significantly(P< 0.05).The level of blood lipid in KK-Ay mice increased,fasting insulin level(INS),HOMA-IR and insulin secretion basic index HOMA-beta increased(P< 0.05).But there was no significant change in the blood lipid of STZ model rats.5.OCT imaging showed that in the 6 months after DM onset,the thickness of retina in STZ rats and KK-Ay mice was significantly reduced compared with C57 mice in normal group.6.The results of HE staining showed that both STZ model 1 DM mice and KK-Ay type 2 DM mice were represented by KK-Ay,and the thickness of the inner layer of the retina decreased significantly compared with the normal group of C57 mice for 6 months.7.Compared with C57 mice in normal control group,the expression level of VEGI protein in STZ and KK-Ay mice retina tissues was significantly reduced(P< 0.05).But there was no statistical difference between STZ and KK-Ay mice.Compared with C57 mice in normal control group,the expression level of VEGF protein in STZ and KK-Ay mice retina tissues increased significantly(P < 0.05).But there was no statistical difference between STZ and KK-Ay mice.Compared with C57 mice in normal control group,the ratio of pAKT/tAKT in retinal tissue of STZ rats and KK-Ay mice increased significantly(P< 0.05).8.In the high glucose state of 30 mM and 50 mM,the proliferation,migration and formation of vascular endothelial cells in bovine retinal vascular endothelial cells were significantly increased,and the proliferation,migration and formation of vascular endothelial cells in bovine retinal vascular endothelial cells were obviously suppressed after VEGI transfection.9.HE staining of the retina tissue section of normal mice showed that the inner boundary membrane was smooth and smooth,without abnormal structure.All the layers of the retina were clearly displayed,and few of the endothelial cells broke through the inner boundary membrane.In the model group,a large number of endothelial cells were directed towards the vitreous cavity,arranged in disorder and neovascularization.In the VEGI intervention group,the number of endothelial cells in the inner limiting membrane of the retina in mice was less than that in the model group,and the integrity of the inner limiting membrane was also good.10.The results of fluorescein perfusion and retina spread showed that the normal mice could be seen radially from the optic disc,distributed evenly and naturally,and did not observe the obvious non perfusion area and the leaking contrast agent.In the model group,a large instillation area was observed around the optic papilla.The large vessels of the retina were irregular dilation,tortuous,and new vascular plexus appeared around the instillation area with obvious fluorescent leakage.In the VEGI intervention group,there was no obvious perfusion area and neovascular plexus around the optic disc,and the fluorescence leakage was alleviated.Conclusions1.Compared with NDM and NPDR patients,the level of VEGI in vitreous decreased and VEGF level increased in PDR patients,and the ratio of VEGI/VEGF decreased significantly.2.Compared with NPDR patients,the level of VEGI in vitreous of PDR patients decreased,while VEGF level increased,and the ratio of VEGI/VEGF decreased significantly.3.Compared with C57 mice in normal control group,the thickness of inner retinal layer in retinal tissue of STZ mice and KK-Ay mice was significantly decreased.The expression level of VEGI protein showed significant decrease,the expression level of VEGF protein increased obviously,the ratio of pAKT/tAKT increased significantly,and the expression level of VEGFR1 protein decreased significantly,while the expression level of VEGFR2 protein increased significantly.4.High glucose can promote the proliferation,migration and formation of bovine retinal vascular endothelial cells,and increase the expression of VEGF in bovine retinal vascular endothelial cells.The glucose effect of 50 mM is more obvious.After transfection,VEGI significantly inhibited the proliferation,migration and tube formation of endothelial cells.5.In hyperoxia induced mice,there were a large number of endothelial cells projecting into vitreous cavity,arranged in disorder,accompanied by neovascularization.The number of endothelial cells that break through the inner limiting membrane in the VEGI intervention group is less than that in the model group,and the integrity of the inner limiting membrane is also good.6.In the hyperoxic induced model group,a large instillation area appeared around the papilla of the mice,and the neovascular plexus appeared around the instillation area with a clear fluorescence leakage.In the VEGI intervention group,there was no obvious perfusion area and neovascular plexus around the optic papilla,and the fluorescence leakage was alleviated.