| BackgroundIschemic retinopathy is a common case,which mainly contains retinopathy Of prematurity(ROP),venous occlusion(RVO)and diabetes mellitus(DR).It is a kind of disease in many countries,and it is the risk and hidden danger of blindness.The retina is the most metabolically active tissue in the body,when the related oxygen consumption is not satisfied in metabolism,it will produce this kind of corresponding disease.For our office workers,DR is a very common cause of blindness.More than 40%of diabetic patients have DR.There will be 366 million people with diabetes and more than 146 million with DR in 2030.The second most common retinal vascular disease is RVO,with a worldwide prevalence estimated at 0.8 per 1,000 people,or about 2.5 million people affected by central retinal vein occlusion,based on a pooled analysis of 15 populations.Severe ROP can lead to blindness.It is currently the number one cause of blindness among children in our country,which imposes a heavy burden on the family and society.In addition to the treatment of the primary disease,the current drug injection treatment methods for ischemic retinopathy are mainly retinal laser and intravitreal injection of anti-VEGF drugs(Ranibizumab,Conbercept,Aflibercept,etc.).Although retinal laser is effective but can lead to retinal photoreceptor function damage,anti-VEGF therapy has been increasingly used in clinical applications in recent years and has achieved good efficacy,but the temporary nature of its effect,the economic burden caused by repeated injections and the atrophy and fibrosis caused by the lack of nutrition of blood vessels and nerves due to repeated injections are still not well resolved.Ischemic retinal disease has not been cured fundamentally.In order to better understand the complex pathological processes of these diseases,large-scale protein studies using high-throughput proteomics techniques have effectively generated important knowledge about the underlying complex pathological mechanisms of RVO,DR and AMD.Proteomics technology has great potential in elucidating the biological processes of the vitreous and retina that occur after treatment with anti-VEGF agents.So far,the proteome changes after intravitreal anti-VEGF treatment have been performed with anti-VEGF drugs bevacizumab and ranibizumab.But there are not many studies on Conbercept.Proliferative diabetic retinopathy(PDR)is the focus of this research.So far,there is no perfect animal model of DR.DR induced by streptozotocin or genetic models simulates the early manifestations of diabetic retinopathy such as leukocyte adhesion,capillary shedding and pericyte loss,but the model cannot enter the proliferation stage.Therefore,other animal models resulting in proliferative retinopathy are commonly used to study PDR in humans.Although the oxygen-induced retinopathy(OIR)model does not have the metabolic alterations of hyperglycemia,it has retinal neovascularization,the most critical feature of human PDR lesions,and is therefore one of the most commonly used models for proliferative retinopathy.Most importantly,whether in the OIR model or in human PDR,early blood vessel loss can lead to pathological new blood vessel formation.The destruction of the blood-retinal barrier and plasma leakage,as well as the neuronal damage,glial and vascular diseases related to vascular diseases can be observed.In this study,an OIR model was used to study the proteomic changes after intravitreal anti-VEGF treatment(Conbercept)for retinal neovascular disease,as a means to screen and identify inflammatory or neovascular factors that remain incompletely suppressed after anti-VEGF treatment for neovascular retinopathy,and to further validate the role and possible mechanisms of this factor and its antagonists in an animal model of OIR.To verify the presence of this factor in the vitreous of patients with proliferative diabetic retinopathy.To investigate the inhibitory effects of antagonists of this factor on vascular endothelial cells in hypoxic and high-glucose environments.Part Ⅰ:Proteomic analysis of Conbercept in the treatment of oxygen induced retinopathy and verification of important protein in animal models and clinical specimensObjectivesTo screen for molecules and associated proteins in neovascular retinopathy that may not be completely inhibited by anti-VEGF treatment by analysing retinal proteomic differences in OIR mice with and without Conbercept treatment.To verify the expression of the calcium-binding protein S100A9 in OIR mice and to validate the therapeutic effect of the S100A9 protein inhibitor Tasquinimod in OIR mice.Verify the expression of S100A9 protein by collecting vitreous from non-diabetic patients compared to proliferative diabetic retinopathy patients.By analyzing the differences in the retinal proteome of oxygen induced retinopathy mice with or without Conbercept treatment,the related proteins in proliferative retinopathy that may not be completely inhibited by anti-VEGF treatment were identified.The expression of the relevant proteins was verified by collecting vitreous from non-diabetic patients compared with patients with proliferative diabetic retinopathy.MethodsC57BL/6J mice postnatal day P7 received 75%oxygen and returned to 21%oxygen environment on P12 to establish an oxygen-induced retinopathy(OIR)model.Collect mouse retinas at P12d(EXP1)and P17d(Control).Conbercept was used to treat retinal neovascularization by intravitreal injection on day P12,and the mouse retinas(EXP2)on day P17 were collected in this group.Retinal whole-mount stained for endothelial cells with isolectin B4 were used to measure the area of non-perfusion area of retina at each time point and each group to observe the effectiveness of treatment.Collect the retinas of each group and each time point,label all the peptides obtained through protein purification,trypsin digestion,Tandem Mass Tag(TMT)reagent,high performance liquid chromatography fractionation,liquid chromatography-mass spectrometry tandem analysis to obtain differential protein data.The secondary mass spectrometry data obtained were searched by Maxquant within the Swiss Prot mouse database and protein functional analysis was performed to screen for the meaningful target calcium binding protein S100A9.For the S100A9 protein antagonist Tasquinimod,OIR mice were divided into OIR+Tasquinimod group with oral Tas(1Omg/KG/d)by gavage starting at P12d,and OIR+PBS group with oral PBS(10mg/KG/d)by gavage starting at P12d.Normal rats and untreated OIR rats were used as controls.P17d the retinas of the above four groups of mice were collected and the inhibitory effect of Tas on the non-perfused areas of the retina was examined by whole mount retinal staining.The expression of S100A9,VEGF-A,intercellular cell adhesion molecule-1(ICAM-1)and thrombospondin-1(TSP-1)in retinal tissues was detected by qPCR and Western blot.The concentration of S100A9 protein detected by the ELISA method was statistically analysed by collecting vitreous humour from non-diabetic patients compared to proliferative diabetic retinopathy patients.ResultsThe retinas of the mice in the EXP1 and Control groups showed obvious retinal non-perfused areas,and the retinal non-perfused areas of the mice in the EXP2 group treated with Conbercept were significantly less than those in the untreated Control group,and the difference was statistically significant(P<0.05).In the course of this project,6701 proteins were obtained,including 5929 quantitative data in quantification.The protein included in bioinformatics analysis was subjected to 1.3-fold change threshold and t-test(P<0.05).In the Control/EXP1 group,365 proteins were up-regulated and 320 proteins were down regulated;in the EXP2/Control group,73 proteins were up-regulated and 63 proteins were down regulated;in the EXP2/EXP1 group,362 proteins were up-regulated and 299 proteins were down regulated.Based on the above data,we conducted bioinformatics analysis of differential proteins.Bioinformatics analysis shows that the KEGG pathways involved in the upregulation of differential proteins during disease progression(Control/EXP1)are mainly phototransduction,ECM-receptor interaction,focal adhesion,glycolysis/gluconeogenesis,HIF-1 signaling pathway,complement and coagulation cascade,etc.The protein domains enrichment involved are EF hand domain,calponin homology domains,epidermal growth factor like domains,etc.The pathways involved in the down-regulation of differential proteins include ribosomal pathway,biosynthetic pathway of terpene backbone,etc.The protein structural domains involved are mini-chromosome maintenance proteins domain,zinc finger protein,and helicase superfamily,etc.Compared to the untreated group(EXP2/Control),up-regulated differential proteins involved in complement and coagulation cascade,lysosomal and apoptosis-related pathways,etc.The protein structural domains involved were β/γ crystal protein,peptidase CIA,papain C-terminus and S100/CaBP-9k type,calcium-binding substructure domain related.The main pathways involved in down-regulation of differential proteins are ribosomes,ECM-receptor interaction,and the protein structural domains involved are linker histone H1/H5,domain H15,histone folding,and histone structural domains.After treatment,compared with the initial stage of treatment(EXP2/EXP1),there are 15 kinds of proteins that are more than 3 times higher.Among them,the calcium-binding protein S100 family contains three,namely:S100A11 protein increased 3.107 times,S100A4 protein increased 3.113 times,S100A9 protein increased 9.06 times.Therefore,S100A9 protein was selected as the next protein for animal and clinical validation.Retinal pavement results after intervention with the S100A9 antagonist Tasquinimod in OIR mice showed that Tas significantly reduced the area of the retinal non-perfused zone in OIR mice.Realtime PCR and Western blot assays showed that oral administration of Tas significantly reduced S100A9,VEGF-A,ICAM-1 and TSP-1 in retinal tissues compared to non-intervened and orally PBS-administered OIR mice.ELISA showed that the concentration of S100A9 protein was significantly higher in the vitreous of proliferative diabetic retinopathy than in non-diabetic patients(patients with idiopathic pre-macular or macular fissure),and the difference was statistically significant.ConclusionsThe S100A9 protein is associated with oxygen-induced retinopathy and its inhibitor Tasquinimod reduces the area of the retinal nonperfused zone in OIR mice by a mechanism that may be related to reduced expression of S100A9,VEGF-A,ICAM-1,and TSP-1 in retinal tissue.S100A9 is highly expressed in the vitreous of patients with proliferative diabetic retinopathy.PART Ⅱ:Effects of S100A9 antagonist Tasquinimod on hypoxia and high-glucose induced human retinal vascular endothelial cellsObjectivesTo investigate the expression of S100A9 in human retinal capillary endothelial cells under Cocl2-induced hypoxia and the inhibitory effect of Tasquinimod,an S100A9 inhibitor,on the migration and vascular network formation of human retinal capillary endothelial cells under hypoxic conditions and the possible mechanisms.To investigate the effects of high glucose on the promotion of S100A9 expression in human retinal capillary endothelial cells and the inhibitory effects of Tasquinimod,an S100A9 inhibitor,on the migration and vascular network formation of human retinal capillary endothelial cells under high glucose conditions,as well as the possible mechanisms.MethodsHuman retinal capillary endothelial cells were divided into normal group(Normal)and Cocl2-induced hypoxia group(Hypoxia)after 24 hours of incubation,and the gene and protein of the cells were extracted for analysis,and the expression of HIF-lα、S100A9 gene and protein of the two groups of cells were detected.Human retinal vascular endothelial cells were divided into different intervention methods:Normal group(Normal),Hypoxia group(200μMCocl2),Hypoxia+1μM Tasquinimod,Hypoxia+5 μM Tasquinimod.The effect of different concentrations of Tasquinimod on the migration of endothelial cells in hypoxic environment was examined by Transwell method;the effect of different concentrations of Tasquinimod on the formation of vascular network of endothelial cells in hypoxic environment was examined by Martrigel endothelial cell lumen forming method.The effects of different concentrations of Tasquinimod on endothelial cell-associated cytokines and channel proteins under hypoxic conditions were examined by Realtime PCR and Western blot.Human retinal vascular endothelial cells were divided into two groups:5mM glucose and 30mM glucose,and after 24 hours of incubation,genes and proteins were extracted from the cells for analysis to detect the expression of S100A9 gene and protein in endothelial cells under different glucose concentrations.The human retinal vascular endothelial cells were divided into four groups:5 mM glucose group,30 mM glucose group,30 mM glucose+10μM Tasquinimod group and 30 mM glucose+50 μM Tasquinimod.The effect of different concentrations of Tasquinimod on the migration of endothelial cells in high glucose environment was examined by Transwell method;the effect of different concentrations of Tasquinimod on the formation of vascular network of endothelial cells in high glucose environment was examined by martrigel endothelial cell lumen formation method.Realtime PCR and Western blot were used to examine the effects of different concentrations of Tasquinimod on endothelial cell-related cytokines and channel proteins in a high glucose environment.ResultsThe gene and protein expression of S100A9 was low in human retinal endothelial cells under normal oxygen concentration,and increased under 200 μM Cocl2-induced hypoxic conditions with statistically significant differences(P<0.05).The migration of cells was inhibited by the addition of 1 μM Tasquinimod and further inhibited by the addition of 5 μM Tasquinimod(P<0.05).In the lumen formation assay,comparison of the branching nodes and length of tubules showed that lumen formation was significantly promoted under hypoxic conditions compared to the normal group,and lumen formation was inhibited by the addition of 1μM Tasquinimod(P<0.05)and further inhibited by the addition of 5μM Tasquinimod(P<0.05).The gene expression of HRECs in the normal and hypoxic groups at different Tasquinimod concentrations was detected by qPCR,which showed that the gene expression of HIF-1α、VEGF-A,ICAM-1 and TSP-1 was significantly promoted in the hypoxic group compared to the normal group.The addition of 1 μM Tasquinimod significantly inhibited the gene expression of HIF-1α、VEGF-A,ICAM-1 and TSP-1 induced by hypoxic stimulation;the addition of 5 μM Tasquinimod further significantly inhibited the gene expression of HIF-1α、VEGF-A,ICAM-1 and TSP-1 induced by hypoxic stimulation,and the difference was statistically significant(P<0.05).The expression of angiogenesis-related cytokines in HRECs at different Tasquinimod concentrations in the normal and hypoxic groups was detected by Western blot,and the hypoxic conditions significantly promoted the expression of HIF-1α、VEGF-A,ICAM-1,TSP-1 and ERK1/2 compared to the normal group.The addition of 1μM Tasquinimod and 5 μM Tasquinimod significantly inhibited the protein expression of HIF-1α、VEGF-A,ICAM-1,TSP-1 and ERK1/2 induced after hypoxic stimulation,with the addition of 5 μM Tasquinimod being more effective(P<0.05).The gene and protein expression of S100A9 was low in human retinal endothelial cells at 5mM glucose concentration and increased at 30mM glucose concentration,the difference was statistically significant(P<0.05).In the Transwell assay,comparison of the number of cells migrating showed that 30 mM glucose significantly promoted cell migration compared to 5 mM glucose,while cell migration was inhibited by the addition of 10 μM Tasquinimod and further inhibited by the addition of 50 μM Tasquinimod(P<0.05).In the lumen formation assay,the comparison of junction nodes and branching length showed that 30 mM glucose significantly promoted lumen formation compared to 5 mM glucose,and the addition of 10μM Tasquinimod inhibited lumen formation(P<0.05),while the addition of 50 μM Tasquinimod further inhibited lumen formation(P<0.05).The gene expression of HRECs at different glucose concentrations and different Tasquinimod concentrations was detected by qPCR,and it was found that 30 mM glucose significantly promoted the gene expression of VEGF and ICAM-1 and inhibited the gene expression of TSP-1 compared with 5 mM glucose,and the difference was statistically significant(P<0.05).The addition of 10 μM Tasquinimod significantly promoted the gene expression of TSP-1 inhibited by high glucose and inhibited the gene expression of VEGF induced by high glucose stimulation;the addition of 50 μM Tasquinimod significantly promoted the gene expression of TSP-1 inhibited by high glucose and inhibited the gene expression of VEGF and ICAM-1 induced by high glucose stimulation.The differences were statistically significant(P<0.05).The protein expression of angiogenesis-related cytokines in HRECs at different glucose concentrations and different Tasquinimod concentrations was detected by Western blot method,and it was found that 30 mM glucose significantly promoted the protein expression of VEGF,ICAM-1 and ERK1/2 and inhibited the protein expression of TSP-1 compared with 5 mM glucose(P<0.05).The addition of 10μM Tasquinimod and 50μM Tasquinimod significantly inhibited the protein expression of VEGF,ICAM-1 and ERK1/2 induced by high glucose stimulation,and promoted the protein expression of TSP-1 inhibited by high glucose,with the addition of 50μM Tasquinimod being more effective(P<0.05).ConclusionsHypoxia and high glucose environment can promote S100A9 expression in endothelial cells,and Tasquinimod,an antagonist of S100A9,can inhibit angiogenesis in hypoxic and high glucose environment by a mechanism that may be related to VEGF-A,ICAM-1,TSP-1,and ERK1/2. |
PDF Full Text Request