Extracellular Vesicle-packaged MiR-143-3p As A Molecular Marker And Therapeutic Target For Intracerebral Hemorrhage

Intracerebral hemorrhage（ICH）is a subtype of stroke with high mortality and disability rates.The condition and prognosis of ICH patients vary greatly among individuals.Due to the lack of reliable biomarkers,it is difficult to identify high-risk patients and provide early intervention.Moreover,the primary hematoma often triggers a cascade of secondary injury responses,leading to continuous disease progression and poor prognosis even after surgical removal of the primary injury.Therefore,there is an urgent need to identify reliable biomarkers for early diagnosis and prediction of disease progression in ICH.Additionally,it is crucial to further elucidate the mechanisms of secondary injury after ICH in order to develop effective targeted treatment strategies and improve patient outcomes.Extracellular vesicles（EVs）are nanoscale vesicles derived from cells.As molecular markers,EVs have many inherent advantages.Firstly,the double-layered membrane structure of EVs protects their contents from degradation,providing stability.Secondly,the contents of EVs are selectively enriched,and their levels or types may have direct relevance to the pathological processes of diseases,making them highly specific.Moreover,EVs are present in various body fluid samples,which are easily collected and provide favorable conditions for timely monitoring of changes in molecular markers during disease progression.In addition to serving as molecular markers,EVs are also important intervention targets for disease treatment.EVs regulate the biological activity of recipient cells through the bioactive lipids,non-coding RNAs,m RNAs,and proteins they carry,playing a significant role in the occurrence and progression of various neurological disorders.Importantly,EVs,as lipid-enclosed vesicles secreted by cells,possess natural drug delivery capabilities.By loading drugs into EVs,advantages such as enhanced stability,improved targeting,and reduced toxicity and side effects can be achieved.In this study,through miRNA sequencing analysis of EVs in plasma samples from ICH patients,we found that the level of miR-143-3p（a brain tissue-specific miRNA）in EVs is closely associated with the neurological functional prognosis of patients.Further basic experiments revealed that EVs-miR-143-3p is mainly derived from astrocytes and can be transferred to brain microvascular endothelial cells（BMECs）,leading to increased expression of inflammatory cell adhesion molecules（CAMs）on BMECs,thereby enhancing the adhesion of peripheral neutrophils to BMECs and promoting neutrophil transendothelial migration（TEM）into the brain,exacerbating secondary injury after ICH.Mechanistically,we found that miR-143-3p can target ATP6V1A（ATPase H+transporting V1 subunit A）,leading to impaired protein autophagy-lysosome degradation pathway and increased expression of CAMs.We constructed an engineered EVs drug delivery system modified with VCAM-1-targeting peptide,which can deliver miR-143-3p inhibitor specifically to inflammatory BMECs,thereby inhibiting the expression of CAMs and neutrophil TEM,and alleviating secondary injury after ICH.In conclusion,our study not only identified EVs-miR-143-3p as a molecular marker for predicting the prognosis of ICH patients but also revealed the important role of EVs-miR-143-3p in the disruption of BMEC barrier function and neutrophil infiltration after acute brain injury.Furthermore,we developed an engineered EVs-based nucleic acid drug delivery system targeting inflammatory BMECs,providing a new approach for the monitoring and treatment of ICH patients in clinical practice.This study is divided into the following three parts:1.Clinical study on EVs-miR-143-3p as a biomarker in ICH patients2.Mechanism study of EVs-miR-143-3p in secondary injury after ICH3.Application study of engineered EVs for targeted delivery of miR-143-3p inhibitor in ICH treatment.Part 1:Clinical study of EVs-miR-143-3p as a biomarker in ICH patientsMethodsIn the screening phase,plasma samples were collected from 25 ICH patients and 20age-matched healthy volunteers.EVs were extracted from plasma using ultracentrifugation,and their successful extraction was confirmed by electron microscopy,size analysis,and immunoblotting.Differential EVs-miRNAs were screened using miRNA-seq.In the validation phase,76 ICH patients and 45 age-matched healthy volunteers were recruited.The expression of differential EVs-miRNAs in plasma was validated by PCR,and multiple logistic regression analysis was conducted to determine the EVs-miRNAs associated with6-month neurological function prognosis（modified Rankin score）in patients.Results1.Analysis of electron microscopy,size analysis,and immunoblotting confirmed the successful extraction of EVs with typical extracellular vesicle structures and marker protein expression.2.Results from miRNA-seq and PCR validation indicated significant differences in the expression of 9 EVs-miRNAs,including EVs-miR-143-3p,after ICH.3.Multiple logistic regression analysis revealed that EVs-miR-143-3p is an independent risk factor for 6-month neurological function prognosis in patients.4.ROC curve analysis demonstrated that EVs-miR-143-3p can effectively predict neurological function prognosis in patients.ConclusionEVs-miR-143-3p is significantly associated with neurological function prognosis in ICH patients.Part 2:Mechanism study of EVs-miR-143-3p in secondary injury after ICHSection 1:EVs-miR-143-3p is mainly secreted by astrocytes after ICH and can lead to dysfunction of brain microvascular endothelial cells.MethodsPrevious studies have reported miR-143-3p as a brain-specific miRNA.To further clarify the neuronal cell sources that contribute to the increased levels of EVs-miR-143-3p after ICH,primary mouse neurons,astrocytes,and microglia,as well as corresponding human neuronal cell lines,were cultured.An in vitro ICH model was established by stimulating the cells with oxyhemoglobin（Oxy Hb）,and the changes in EVs-miR-143-3p levels secreted by different types of neuronal cells before and after injury were detected using PCR.To determine if astrocytes are the main source of EVs-miR-143-3p after ICH,the expression of SMPD3,a specific gene in astrocytes involved in EVs synthesis,was knocked down in mice.Changes in EVs-miR-143-3p in brain tissue were then examined.To investigate whether brain microvascular endothelial cells can uptake EVs-miR-143-3p,the uptake of EVs derived from human astrocytes（NHA）by human brain microvascular endothelial cells（h CMEC/D3）was validated in vitro.Changes in miR-143-3p levels in brain microvascular endothelial cells before and after ICH were also examined after specific knockdown of SMPD3 in astrocytes in vivo.The effects of miR-143-3p on endothelial cells were studied by transfecting h CMEC/D3 cells with miR-143-3p mimics,analyzing apoptosis using flow cytometry,and examining the expression of proteins related to endothelial cell barrier function.Furthermore,the changes in the barrier function of brain microvascular endothelial cells were evaluated after stereotactic injection of GFAP-AAV-miR-143-3p sponge,specifically targeting astrocytes,in ICH mice to inhibit the function of miR-143-3p.Results1.The levels of EVs-miR-143-3p in mouse plasma and brain tissue significantly increased after ICH.2.After Oxy Hb treatment,the secretion of EVs-miR-143-3p increased significantly in primary mouse astrocytes and human astrocyte cell lines,but not in microglia or neurons.3.Knockdown of SMPD3 in astrocytes did not significantly affect the levels of EVs-miR-143-3p in brain tissue before and after ICH.4.After ICH,the level of miR-143-3p in brain microvascular endothelial cells significantly increased,but this elevation was not observed after specific knockdown of SMPD3 in astrocytes.5.miR-143-3p did not affect the viability of h CMEC/D3 cells but significantly increased the expression of cell adhesion molecules（CAMs）associated with inflammatory response.6.Inhibition of miR-143-3p function by GFAP-AAV-miR-143-3p sponge injection in astrocytes did not lead to a significant increase in CAMs expression in brain microvascular endothelial cells after ICH.ConclusionAfter ICH,astrocytes secrete EVs-miR-143-3p,which promotes the expression of CAMs in brain microvascular endothelial cells.Section 2:miR-143-3p inhibits CAMs autophagic degradation by targeting ATP6V1AMethodsqPCR was used to detect the effect of miR-143-3p on CAMs m RNA levels.Protein degradation pathways were assessed using the proteasome inhibitor MG132 and the lysosome inhibitor Baf A1 in endothelial cells.WB was used to detect autophagy markers SQSTM1 and LC3,and cells were transfected with m RFP-GFP-MAP1LC3B adenovirus to assess the effect of miR-143-3p on autophagic flux.Target genes potentially regulated by miR-143-3p were predicted using Target Scan,miRanda,miRDB,and miRWalk software.Luciferase and WB experiments were conducted to validate the regulation of target genes by miR-143-3p.The effect of miR-143-3p on lysosomal function and CAMs expression was investigated in h CMEC/D3 cells treated with the ATP6V1A activator EN6.Results1.miR-143-3p did not significantly affect the m RNA levels of VCAM-1,ICAM-1,and PECAM-1 in endothelial cells.2.Bioinformatics analysis did not reveal direct binding sites between miR-143-3p and the 3’-UTR regions of VCAM-1,ICAM-1,and PECAM-1.3.Inhibition of the autophagy-lysosome pathway increased the protein levels of VCAM-1,ICAM-1,and PECAM-1 in h CMEC/D3 cells,while inhibition of the ubiquitination-proteasome pathway had no significant effect.4.miR-143-3p inhibited lysosomal degradation of autophagosomes in h CMEC/D3 cells.5.miR-143-3p directly regulated ATP6V1A,a key protein involved in lysosomal H+transport and acidification.6.miR-143-3p increased lysosomal p H and decreased the activity of lysosomal proteases CTSB and CTSD in h CMEC/D3 cells.7.Activation of ATP6V1A reversed the increase in lysosomal p H,impairment of lysosomal hydrolytic function,and upregulation of CAMs expression caused by miR-143-3p.ConclusionmiR-143-3p inhibits autophagic degradation of CAMs in endothelial cells by targeting ATP6V1A.Section 3:Endothelial cell-specific deletion of Atp6v1a exacerbates CAMs expression and neutrophil infiltration in ICH-induced secondary injuryMethodsWe generated endothelial cell-specific Atp6v1a knockout mice(Atp6v1a^CKO mice)and assessed the effect of Atp6v1a deletion on CAMs expression after ICH using WB.CAMs are key molecules that mediate adhesion between circulating immune cells and brain microvascular endothelial cells（BMECs）,promoting their transendothelial migration.Therefore,we further used flow cytometry to quantify different immune cell populations,including neutrophils,T cells,macrophages/monocytes,and NK cells in the brain.To evaluate the impact of Atp6v1a deletion on secondary brain injury,we measured the hemorrhage volume using MRI,assessed changes in brain water content using the wet-dry method,and detected neuronal apoptosis using TUNEL staining.Results1.WB results showed a significant decrease in ATP6V1A protein expression in BMECs24 hours after ICH.2.Compared to control mice,Atp6v1a^CKO mice exhibited a more significant increase in CAMs expression levels after ICH.3.Compared to control mice,Atp6v1a^CKO mice showed a more significant increase in neutrophil infiltration 24 hours after ICH,a slight increase in macrophages/monocytes,and no significant increase in T cells and NK cells.4.There was no significant difference in hemorrhage volume between control mice and Atp6v1a^CKO mice 24 hours after ICH.5.Compared to control mice,Atp6v1a^CKO mice exhibited more neuronal apoptosis and more severe brain edema after ICH.ConclusionEndothelial cell-specific deletion of Atp6v1a exacerbates CAMs expression and neutrophil infiltration in ICH-induced secondary injury.Part III:Application of engineered EVs targeting delivery of miR-143-3p inhibitor for alleviating secondary injury after ICHSection 1:Construction of engineered EVs targeting brain microvascular endothelial cells with miR-143-3p inhibitorMethods:Based on the previous studies,we aimed to construct a drug delivery system that can specifically target and deliver miR-143-3p inhibitor to damaged brain microvascular endothelial cells.EVs were extracted from the cell culture medium of 293T cells,and miR-143-3p inhibitor was loaded into the EVs using electroporation（350 V/150 m F）.VCAM-1targeting peptide（VHPKQHR）was engineered to the surface of EVs.The VCAM-1targeted,miR-143-3p inhibitor-loaded engineered extracellular vesicles（Vt-miR-143-3pi-EVs）were characterized using NMR,electron microscopy,size analysis,and Western blot.The targeting and delivery capabilities of Vt-miR-143-3pi-EVs were validated through immunofluorescence analysis in vitro and in vivo imaging system（IVIS）analysis after labeling with fluorescent dyes.Results:1.1H NMR,electron microscopy,size analysis,and WB analysis confirmed the successful construction of Vt-miR-143-3pi-EVs.2.In vitro experiments showed that resting h CMEC/D3 cells had limited uptake of miR-143-3pi-EVs and Vt-miR-143-3pi-EVs.However,TNFα-induced activated h CMEC/D3cells exhibited significantly enhanced uptake of Vt-miR-143-3pi-EVs,while the uptake of non-targeted miR-143-3pi-EVs remained unchanged.3.In vivo imaging experiments demonstrated that Vt-miR-143-3pi-EVs had better accumulation in the damaged brain region compared to miR-143-3pi-EVs.4.Vt-miR-143-3pi-EVs could suppress the expression of ATP6V1A in brain microvascular endothelial cells after ICH.Conclusion:Engineered EVs targeting VCAM-1 can effectively deliver miR-143-3p inhibitor to activated brain microvascular endothelial cells.Section 2:Vt-miR-143-3pi-EVs alleviate secondary injury after ICH and improve functional outcomesMethods:Immediately after ICH induction,200μg of Vt-miR-143-3pi-EVs were administered via tail vein injection once daily.The expression of CAMs（VCAM-1,ICAM-1,and PECAM-1）was measured by WB at 1 day after ICH.Neutrophil infiltration was assessed by flow cytometry,neuronal apoptosis was evaluated using TUNEL staining,and motor function was assessed through the foot fault test,rotarod test,and adhesive removal test in mice.Results:1.Administration of Vt-miR-143-3pi-EVs effectively suppressed the increased expression of VCAM-1,ICAM-1,and PECAM-1 after ICH.2.Administration of Vt-miR-143-3pi-EVs effectively reduced neutrophil infiltration after ICH.3.Administration of Vt-miR-143-3pi-EVs effectively attenuated neuronal apoptosis after ICH.4.Administration of Vt-miR-143-3pi-EVs effectively alleviated brain edema after ICH.5.Administration of Vt-miR-143-3pi-EVs effectively improved neurological deficits after ICH.Conclusion:Vt-miR-143-3pi-EVs can effectively alleviate secondary injury after ICH and promote functional recovery.