Quantitative Proteomic Analysis Of Plasma-derived Exosomes In Patients With Diabetic Retinopathy

Objective: Diabetic retinopathy(DR)is the main cause of blindness,and the pathogenesis of it has not yet been fully elucidated.The protein profile of plasma's exosomes in proliferative diabetic retinopathy(PDR)was analyzed by using proteomics technology to find biomarkers and explore the mechanism and potential therapeutic targets of PDR.Method:1?We collected,identified,and analyzed the plasma exosomes of PDR patients.The clinical information of PDR patients hospitalized in Tianjin Medical University Eye Hospital,patients with diabetes without retinopathy(DM),and normal controls(patients with macular hole,or MH)was collected.The plasma from patients was collected,the plasma-derived exosomes were separated by ultracentrifugation,the morphology and size of exosomes were observed by transmission electron microscope,and the surface biomarkers of exosomes CD9,CD61 and CD81 were verified by Western blot(WB).The protein concentrations of exosomes and microvesicles(MV)derived from 2ml plasma were measured,respectively,and Coomassie Brilliant Blue assay was used to analyze the protein distribution of exosomes and other components of the plasma.2? We performed quantitative proteomic analysis of plasma-derived exosomes,which was further verified by enzyme-linked immunosorbent assay(ELISA).The exosomes from the 5 patients within each group were pooled,and the concentration for each group was tested by BCA assay;then,equal amounts of each i TRAQ-labelled sample were mixed and underwent quantitative proteomic analysis and data analysis.Screening of the credible proteins and differential proteins,followed by Gene Ontology analysis of the differential proteins,and the differences of plasma-derived exosomes,microvesicles(MVs),extracellular vesicles,free plasma,and plasma were validated by ELISA.The expression of the vitreous proteins derived from PDR patients and normal controls(MH)was simultaneously confirmed by ELISA.The expression of tumor necrosis factor-induced protein in endothelial cells of oxidative stress model were verified by using q-PCR and WB.3?Eestablished a cell model of oxidative stress for protein profiling in retinal microvascular endothelial cells.The oxidative stress model of retinal microvascular endothelial cells(HRMEC)was successfully constructed,then HRMEC was stimulated with 4-hydroxynonenal(4-HNE),and the optimal concentration of 4-HNE to stimulate cell proliferation was detected by CCK-8.Finally,10 ?M 4-HNE was used to stimulate HRMEC,and cell proteins were extracted for SWATH data independent quantitative analysis.Results:1? Analysis of clinical information of patients: There were no significant differences in age,gender,height,weight,or cholesterol,triglyceride,creatinine,urea,nitrogen,and blood pressure levels between groups.The glycosylated hemoglobin in the DR group was slightly higher than the DM group but not statistically significant.2? Exosome identification: Exosome electron microscopy showed double-layered round cakes of tray-like and teacup-like appearance with diameters between 40-100 nm.Western blot showed exosomes contained CD9,CD61,and CD81.3? Coomassie brilliant blue results: Exosome,MV,extracellular vesicle-free plasma and plasma protein distribution differences were shown.The content of exosome protein in the DR group was lower than that in the DM group and the normal group(P<0.05).There was no difference in the protein concentration of MV between the groups.4? Results of plasma exosome proteomics: A total of 856 proteins were detected in the exosome protein profile,of which 5 were upregulated in PDR compared with DM and normal,tumor necrosis factor-induced protein 8(TNFAIP8),protein S100-A8,protein S100-A9,zinc finger protein 736 and plasma glutathione peroxidase;179 proteins had reduced expression compared with DM and normal protein,including breast cancer type 1 susceptibility protein and neurogranin.The most significantly downregulated proteins were megakaryocyte and platelet inhibitory receptor G6 b.34 proteins were downregulated in the DM group.5? ELISA verification results: TNFAIP8 expression in plasma exosomes of PDR patients was higher than the DM and normal groups.Vitreous showed the same trend,and the mass spectrometry results are the same and statistically significant.There was no difference in the expression of the three groups MV,free plasma,and plasma;the content was low.ELISA results of breast cancer type 1 susceptibility protein and neurogranin showed no difference in exosome protein expression in each group.6? Construction of oxidative stress model: The CCK-8 experiment showed that different 4-HNE concentrations had different effects on HRMEC activity,and 10?M 4-HNE had the most obvious proliferation of HRMECs.7? Expression of TNFAIP8 in oxidative stress model: q-PCR and WB verified the expression of tumor necrosis factor-induced protein in oxidative stress model.8? Quantitative proteomic mass spectrometry of HRMECs found that various proteins changed under oxidative stress conditions,mainly antioxidant stress protein,ATPase and programmed cell death protein 4.Conclusion:1?There is a difference in protein composition between plasma-derived exosomes and other components of plasma.2?The expression of TNFAIP8 in plasma exosomes and vitreous is increased in patients with PDR,and the expression level in plasma is low.3?TNFAIP8 may originate from retinal microvascular endothelial cells and enter the systemic blood circulation through plasma exosomes which can be used as a biological marker of DR.4?The proliferation of HRMECs under oxidative stress may be through up-regulation of anti-oxidative stress protein and ATPase and down-regulation of programmed cell death protein 4.