The Role And Mechanism Of Mesenchymal Stem Cell-derived Small Extracellular Vesicles In Diabetic Retinopathy Therapy

Objective:As a microvascular complication of diabetes caused by persistent hyperglycemia,diabetes retinopathy(DR)has been emerged as an important cause of vision decline and blindness in adults.Current treatment strategies provide limited effects.Recently,mesenchymal stem cell-derived small extracellular vesicles(MSC-s EVs)play important roles in various tissue regeneration and are considered as a novel cell-free strategy.This study aims to investigate the intervention effect of MSC-s EVs on hyperglycemia-induced retinal injury,analyze the key molecules in MSC-s EVs and the specific mechanism underlying MSC-s EVsmediated retinal therapy,establish engineering methods to modify MSC-s EVs for improving the therapeutic effects,and provide a novel approach for DR therapy.Methods:(1)The primary MSC was isolated by the adherent method of umbilical cord tissue.The morphological characteristics of MSC were observed under the microscope.After adipogenic and osteogenic induction,Oil-Red-O and Alizarin-Red-S staining were performed to identify the multidirectional differentiation ability of MSC.The surface marker expression of MSC was detected by flow cytometry.MSC-s EVs were isolated from the conditioned culture supernatant of MSC by ultracentrifugation.The morphology and structure of MSC-s EVs were detected by transmission electron microscopy.The particle size and concentration of MSC-s EVs were detected by nanoparticle tracking analysis.The protein marker expression of MSC-s EVs was determined by Western blot.(2)Two DR animal models were constructed,including db/db mice and streptozotocin(STZ)-induced DR rat model,followed by the intravitreal injection of MSC-s EVs.The effect of MSC-s EVs on retinal function was detected by electroretinography analysis.The effect of MSC-s EVs on retinal tissue structure was observed by hematoxylin and eosin(H&E)staining of retinal sections.The effect of MSC-s EVs on retinal apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling(TUNEL)assay,immunohistochemistry staining and Western blot.The retinal expression level of inflammatory factors after MSC-s EVs intervention was detected by quantitative real-time polymerase chain reaction(q RT-PCR).The effect of MSC-s EVs on retinal pathological angiogenesis was determined by trypsin digestion assay,Evans blue leakage assay,immunohistochemical staining and Western blot.The retinal therapeutic effect of MSC-s EVs on DR animal models was evaluated according to the changes of above indexes.Human retinal microvascular endothelial cells(h RMECs)were stimulated with high glucose medium and then treated with MSC-s EVs in vitro.CCK8 assay and Ed U staining were adopted to determine the proliferation potential of h RMECs.Transwell assay was adopted to investigate the migration potential of h RMECs.Tube formation assay was used to determine the angiogenesis potential of h RMECs.The intervention effect of MSC-s EVs on h RMECs was evaluated according to the changes of cellular function.(3)The differentially expressed genes in retinal tissues after MSC-s EVs treatment were analyzed by transcriptome sequencing,and the potential target of MSC-s EVs was identified.The regulatory effect of MSC-s EVs on this target was verified by immunofluorescence staining and Western blot.Mass spectrometry,immunofluorescence staining,Western blot,Co-immunoprecipitation(Co-IP)assay and q RT-PCR were used to explore the specific mechanism of this target to participate in DR progress.(4)Mi RNA sequencing was used to identify the mi RNA expression profile of MSCs EVs and screen the mi RNA molecules that were abundant in MSC-s EVs and predicted to target the therapeutic target.The binding site was predicted by bioinformatics software.After the synthesis of wild-type and mutant plasmids and mi RNA mimics,the plasmids and mi RNA mimics were co-transfected into human embryonic kidney 293T(HEK293T)cells,followed by the dual-luciferase reporter gene analysis to detect the binding between mi RNA and target m RNA.The effect of mi RNA mimics and inhibitors transfection on the function of h RMECs was detected by CCK8 assay,Ed U staining,Transwell assay and tube formation assay.The mi RNA inhibitors were introduced into MSC-s EVs by electroporation to inhibit the mi RNA function,and then db/db mice were treated with these MSC-s EVs.The retinal function,structure,apoptosis,inflammation and angiogenesis were detected to determine whether the screened mi RNA mediated the retinal repairing effects of MSC-s EVs.(5)The exogenous mi R-5068 and mi R-10228 mimics were loaded into MSC-s EVs by electroporation to construct engineered MSC-s EVs.The level of key mi RNA in engineered MSC-s EVs was detected by q RT-PCR.The morphological structure,particle size and protein marker expression of engineered MSC-s EVs were identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot.The retinal therapeutic effect of engineered MSC-s EVs on DR animal model was assessed by detecting the retinal function,structure,apoptosis,inflammation and pathological angiogenesis.The biological safety of engineered MSC-s EVs was evaluated by the H&E staining of major organs and serological indicators of liver and kidney function.Results:(1)MSC isolated from the umbilical cord tissue exhibited long spindle morphology and could differentiate into adipocytes and osteoblasts.MSC positively expressed CD29,CD73 and CD105,and negatively expressed CD11 b,CD34 and CD45.MSC-s EVs were successfully isolated by ultracentrifugation.MSC-s EVs exhibited spherical vesicles with the particle size of 163.1nm.MSC-s EVs were positive for the expression of protein markers such as tumor susceptibility gene 101(TSG101),Alix,CD9 and CD63,and negative for the expression of Calnexin.(2)The DR animal models were successfully established.Compared with the normal group,the model group showed degenerated retinal function,disordered retinal structure,reduced retinal thickness,increased retinal apoptosis,upregulated expression of inflammatory factors and enhanced neovascularization.The intravitreal injection of MSC-s EVs effectively improved retinal function,maintained retinal integrity and increased retinal thickness.MSCs EVs could reduce the number of TUNEL positive cells,alleviate retinal apoptosis and inhibit the release of inflammatory factors including interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β).Moreover,MSC-s EVs downregulated the retinal expression of vascular endothelial growth factor(VEGF),enhanced the tight connection among blood vessels,alleviated the vascular leakage and reduced the number of acellular capillaries.The results in vitro also showed that MSCs EVs could alleviate the high glucose-induced enhanced proliferation,migration and angiogenesis abilities of h RMECs.(3)Transcriptome sequencing results showed that the differentially expressed genes in retinal tissues after MSC-s EVs treatment were enriched in hypoxia-inducible factor-1α(HIF-1α)pathway.In vivo and in vitro experiments indicated that high glucose could cause the upregulation and nuclear transcription of HIF-1α,while MSC-s EVs inhibited HIF-1αexpression.The HIF-1α agonist DMOG significantly weakened the repairing function of MSC-s EVs in h RMECs cultured in high glucose medium.In addition,the results of mass spectrometry,immunofluorescence staining and Western blot revealed that HIF-1αknockdown could promote peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α)expression.Cell function experiments demonstrated that the downregulation of PGC-1αremarkably impaired HIF-1α knockdown-induced the functional recovery of h RMECs.Bisulfite sequencing PCR results showed that HIF-1α knockdown significantly reduced the methylation level in PGC-1α gene promoter.Immunofluorescence staining and Co-IP assay confirmed the binding between HIF-1α and enhancer of zeste homologue 2(EZH2).The knockdown of EZH2 enhanced the expression level of PGC-1α in h RMECs.The results of chromatin immunoprecipitation(Ch IP)assay revealed that EZH2 protein could interact with PGC-1α DNA.The study involving the mechanism responsible for the interaction between HIF-1α and EZH2 showed that the expression change of HIF-1α did not affect EZH2 m RNA level,but altered EZH2 protein expression.The results of proteasome inhibitor MG132 assay and cycloheximide assay showed that HIF-1α regulated EZH2 protein stability through ubiquitin-proteasome pathway.Co-IP assay and mass spectrometry confirmed that HIF-1αinhibited E3 ligase tripartite motif 21(TRIM21)-mediated EZH2 ubiquitination and degradation.(4)The results of mi RNA sequencing and q RT-PCR showed that mi R-5068 and mi R-10228 were abundant in MSC-s EVs.Dual-luciferase reporter gene analysis confirmed that HIF-1α was the target of mi R-5068 and mi R-10228.Transfection with mi R-5068 and mi R-10228 mimics could alleviate the high glucose-induced increased proliferation,migration and angiogenesis of h RMECs.In addition,the inhibition of mi R-5068 and mi R-10228 impaired the MSC-s EVs-mediated regulation on HIF-1α/EZH2/PGC-1α pathway and therapeutic effects to recover retinal function and inhibit retinal apoptosis,inflammation and pathological angiogenesis.(5)Engineered MSC-s EVs constructed by electroporation exhibited increased levels of mi R-5068 and mi R-10228,maintained the spherical-shaped morphology with a slight increase in size and expressed protein markers of EVs.Compared with natural MSC-s EVs,the intravitreal injection of engineered MSC-s EVs further improved retinal function and inhibited retinal apoptosis,inflammation and angiogenesis in db/db mice and STZ-induced DR rats.The H&E staining of main organs and serological examination showed that the intervention of engineered MSC-s EVs did not cause systemic adverse reactions.Conclusions:MSC-s EVs alleviate hyperglycemia-induced retinal injury by improving retinal function and inhibiting retinal apoptosis,inflammation and pathological angiogenesis.Our findings suggest that MSC-s EVs-delivered mi R-5068 and mi R-10228 inhibit HIF-1αexpression,thus promoting the TRIM21-mediated ubiquitination and degradation of EZH2.The downregulation of EZH2 promotes downstream PGC-1α expression by reducing the methylation modification in PGC-1α gene promoter.Mi R-5068 and mi R-10228-engineered MSC-s EVs constructed by electroporation show enhanced retinal repairing effects with biosafety.This study provides new insights into the mechanism of DR progress and supplies a new cell-free strategy for DR treatment.