Histological Analysis Of Vitreous Exosomes In Proliferative Diabetic Retinopathy

Purpose:To evaluate the differences in exosomal protein expression in vitreous humor(VH)of patients with proliferative diabetic retinopathy(PDR)and controls(patients with idiopathic macular hole or idiopathic macular epiretinal membrane)and to explore the role of the exosomal protein in the mechanism of PDR occurrence and development.Methods:A total of 38 patients were enrolled in this study between July 2020 and September 2021 at the Ophthalmology Department of Qilu Hospital,Shandong University,and were divided into a test group and a control group;the test group consisted of patients with PDR(vitrectomy)and the control group consisted of patients with idiopathic macular hole or idiopathic macular epiretinal membrane without diabetes mellitus.The trial was divided into two phases(sequencing phase and validation phase);8 cases(4 subjects in each group)were included in the sequencing phase and 30 cases(20 patients with PDR and 10 controls)in the validation phase.General baseline information such as age,gender,triglycerides(TG),total cholesterol(TCho),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),and other factors were measured in both groups showed no statistical differences.The exosomes were extracted from the vitreous humor by differential ultracentrifugation,and then identified as exosomes by transmission electron microscopy(TEM),nanoparticle tracking analysis techniques(NTA),nano-flow cytometry(NanoCM).The exosomal proteins were identified using Lable-free proteomic analysis combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)to determine the differences in exosomal protein expression between the two groups of patients.Parallel Reaction Monitoring(PRM)was used to detect the expression of differentially expressed exosomal proteins in the validation set of patients.Bioinformatic analysis of the screened differential proteins was performed by gene ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG).The diagnostic value of the differential proteins in distinguishing PDR from non-diabetic controls was evaluated by applying the receiver operating characteristic curve(ROC)and calculating the area under curve(AUC).All statistical analyses were performed using GraphPad Prism(GraphPad Prism,Version 5)and P values<0.05 were considered statistically significant.Results:1.TEM,NTA and NanoFCM confirmed that the isolated extracellular vesicles showed a typical circular disc-like structure with a diameter distribution between 30 and 120 nm and expressed the main characteristic proteins,CD9,CD63 and CD81,and their morphology,size and protein markers were consistent with exosomes.2.Exosomes from vitreous fluid of PDR patients and their controls analysed by Lable-free proteomic analysis identified a total of 758 proteins in the two groups,of which 10 proteins were significantly differentially expressed between the two groups(P<0.05,>2.0-fold change or<0.5-fold change in ploidy).Among them four were up-regulated in the PDR group,including immunoglobulin lambda-like polypeptide 1(IGLL1),triosephosphate isomerase 1(TPI1),lactate dehydrogenase A(LDHA)and apolipoprotein B(ApoB),and six were down-regulated,including ficolin 3(FCN3),aldolase C(ALDOC),serum amyloid protein(SAA)2-4,apolipoprotein M(ApoM),insulin-like growth factor binding protein,acid-labile subunit(IGFALS)and hyaluronan binding protein 2(HAB2).3.GO analysis showed that these differential proteins are mainly involved in various metabolic pathways,including nicotinamide purine dinucleotide metabolism,adenosine diphosphate metabolism and glycolysis.In addition,KEGG analysis suggested that these differentially expressed exosomal proteins were mainly involved in glycolysis and metabolic pathways such as gluconeogenesis,fructose and mannitol metabolism,amino acid biosynthesis,hypoxia-inducible factor 1 signalling pathway and carbon metabolism.4.Further PRM quantitative analysis suggested that the expression levels of LDHA,FCN3,ApoB and ApoM were consistent with the results of proteomics.5.ROC analysis suggested that these four proteins could be used as biomarkers for the diagnosis of PDR(AUC>0.75).Conclusion:PDR patients have a large amount of proteins in their vitreal exosomes,and there are significant differences in the protein expression profiles of vitreal exosomes between PDR patients and control groups.Bioinformatics analysis suggests that these differentially expressed exosomal proteins mainly mediate various glucose and lipid metabolism pathways,which are closely related to the occurrence and development of PDR,and have high efficacy in distinguishing PDR patients from control groups,indicating that these differentially expressed exosomal proteins may participate in the occurrence and development of PDR through exosomal pathways and may serve as novel biomarkers and potential therapeutic targets for PDR.