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Brain endothelial cell-derived extracellular vesicles (c-BEEVs) as a promising biomarker for brain vascular pathology and cognitive decline

Nature Aging (2026) Cite this article

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Abstract

Accurate measurement of brain vascular pathology is essential for understanding its role in cognitive aging. Here we classified participants using the amyloid-tau-neurodegeneration framework in a multicenter cohort and identified cerebrospinal fluid brain endothelial-derived small extracellular vesicles (c-BEEVs) as a sensitive biomarker, which correlated with vascular risk factors and the severity of small-vessel disease. c-BEEVs showed high diagnostic performance for vascular cognitive impairment and, when combined with p-tau181, effectively distinguished vascular cognitive impairment from Alzheimer’s disease. In individuals with mixed Alzheimer’s disease and vascular pathology, c-BEEVs were the earliest indicators of abnormalities. It predicted cognitive decline in participants without p-tau181 pathology. To investigate the mechanistic role of c-BEEVs, we established a hypertension mouse model with elevated c-BEEVs and cognitive deficits. Brain endothelial-specific knockdown of extracellular vesicle secretion alleviated cognitive and synaptic impairment. These findings position c-BEEVs as a promising biomarker for brain vascular pathology and highlight their role in neurovascular dysfunction.

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Fig. 1: Overview of the study.
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Fig. 2: EV biomarkers measured in the discovery and validation cohorts.
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Fig. 3: Diagnostic and differential diagnostic performance of c-BEEVs.
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Fig. 4: Biomarker-based staging and the prognostic value of c-BEEVs.
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Fig. 5: BEEVs mediate HTN-induced cognitive impairment and synaptic dysfunction.
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Fig. 6: Elevated CSF BEEVs promote cognitive impairment in the HTN model.
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Data availability

Experimental data and methods supporting the findings of this study are available within the article and its Supplementary Information. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE99 partner repository with the dataset identifier PXD074721. Raw clinical data are not publicly available because of participant privacy and ongoing follow-up studies but are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code for the SuStaIn model was obtained from the publicly available repository (https://github.com/ucl-pond/pySuStaIn). All other analyses were performed using standard R packages, and no custom code was developed. Additional details are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank the Shu Yousheng Lab at the Institute for Translational Brain Research of Fudan University for providing the platform and guidance to the electrophysiological experiments. This study was supported by the Ministry of Science and Technology of China (STI2030-Major Projects 2030 2021ZD0201806 to M.C., 2021YFC2500100 to Q.D.), Natural Science Foundation of China (82271221 to M.C.), Shanghai Municipal Health Commission (20234Z0013 to M.C.), Shanghai Oriental Talent Program (M.C.), Shanghai Medical New Star Program (M.C., Y.W.), and Clinical Research Special Program for the Health Industry (20244Y0103 to Y.W.).

Author information

Author notes

  1. These authors contributed equally: Tongyao You, Yingzhe Wang, Jun Xu, Yanxin Zhao, Sisi Peng.

Authors and Affiliations

  1. Department of Neurology and National Center for Neurological Disorders, Huashan Hospital, Fudan University, Shanghai, China

    Tongyao You, Yingzhe Wang, Qi Han, Xun Wang, Weiwei Shen, Min Guo, Jintai Yu, Qiang Dong & Mei Cui

  2. Beijing Tiantan Hospital, Capital Medical University, Beijing, China

    Jun Xu

  3. The Second People’s Hospital of Guiyang, Guiyang, China

    Jun Xu

  4. Department of Neurology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China

    Yanxin Zhao & Minjie Zhang

  5. Department of Neurology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China

    Yanxin Zhao

  6. Department of Neuropsychology, Zhongnan Hospital of Wuhan University, Wuhan, China

    Sisi Peng & Junjian Zhang

  7. Carrier Biomed (Suzhou) Company, Suzhou, China

    Hailun Yu

  8. State Key Laboratory of Brain Function and Disorders and MOE Frontiers Center for Brain Science, Shanghai Medical College, Fudan University, Shanghai, China

    Jintai Yu, Qiang Dong & Mei Cui

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  1. Tongyao You
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  2. Yingzhe Wang
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Contributions

M.C., Q.D., and J.Y. designed the study. T.Y., Q.H., Y.W., X.W., J.X., S.P., Y.Z., and J.Z. collected the data. T.Y. analyzed the data, performed the experiments, and drafted the paper. H.Y., M.Z., W.S., and M.G. helped with the experimental methodology. All authors contributed to the interpretation of the results, revised the paper, and approved the final version.

Corresponding authors

Correspondence to Jintai Yu, Qiang Dong or Mei Cui.

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Competing interests

The authors declare no competing interests.

Peer review

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Nature Aging thanks Xi Chen, Dimitrios Kapogiannis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Cross-validation of CD31+ EV detection by nano-flow cytometry.

a Representative nano-flow cytometry plots showing the proportions of CD31+GLUT1+ particles (Quadrant 2) in CSF samples. Cohorts were stratified into lower, middle, and upper tertiles based on c-BEEV levels: [8, 22), [22, 26], and (26, 41]. The double-positive (DP) percentage was calculated as the number of CD31+GLUT1+ particles in Quadrant 2 divided by the total number of CD31+ particles in Quadrants 1 and 2. b Scatter plot showing correlations between CD31+ particles detected by NTA with CD31+ or CD31+ GLUT1+ particles detected by nano-flow cytometry. (n = 14, low tertile; n = 18, middle tertile; n = 23, upper tertile; Pearson’s correlation). c-d Representative nano-flow cytometry plots (c) and quantitative analysis (d) showing the percentages of CD31+ serum EVs isolated from participants in the indicated groups. (CV, n = 40; CAS, n = 39; CSVD, n = 42; One-way ANOVA with Tukey’s correction). e Representative flow cytometry plot of forward scatter versus FITC intensity showing that, in the EV sample captured by CD31-coupled magnetic beads, 99.65% particles were FITC-positive. The degree of flow separation is shown in the right panel. f Representative nano-flow cytometry plots showing the percentages of CD31+ EVs in the CD31+ beads-captured fraction versus the remaining fraction. Data are presented as mean ± SEM. Abbreviations: NTA, nano tracking analysis; CV, community volunteers; CAS, carotid artery stenosis; CSVD, cerebral small vessel disease; EV, extracellular vesicles.

Extended Data Fig. 2 Levels of c-BEEVs across cognitive impairment groups stratified by CDR score.

Participants with available CDR scores were grouped according to their CDR stage. Sample sizes for each group are provided in Supplementary Table 4. Box plots show the median (center line), the interquartile range (box, 25th-75th percentiles), and whiskers extending to 1.5 × the interquartile range. Each dot represents an individual subject. Statistical comparisons were performed using one-way ANOVA with Tukey’s correction. Abbreviations: CDR, clinical dementia rating; c-BEEVs, the percentage of CD31+ small extracellular vesicles among total small extracellular vesicles in the cerebrospinal fluid; AD, Alzheimer’s dementia; VCI, vascular cognitive impairment; MD, mixed dementia.

Extended Data Fig. 3 Vascular injury, blood-brain barrier permeability, and changes in microvessel structure of the hypertension model.

a Top: Representative images of myelin basic protein (MBP) staining in the brains of hypertension (HTN) model mice show partial demyelination. Scale bar, 200 μm. Bottom: Representative images of hematoxylin and eosin (HE) staining in the brains of HTN model mice show enlarged perivascular spaces (ePVS). Scale bar, 20 μm. b Quantification of normalized MBP intensity in the indicated groups (n: the number from 4 mice per group, unpaired two-sided t-test). c Quantification of the mean area percentage of ePVS in the indicated groups (n: the number from 4 mice per group, unpaired two-sided t-test). d Representative images of albumin (magenta, MW 66.5 kDa) and CD31+ microvessels (gray) in the cortex and hippocampus of sham and HTN model mice. Scale bar, 200 μm. e Representative images of fibrinogen (Fib, magenta, MW 340 kDa) and CD31+ microvessels (gray) in the cortex and hippocampus of sham, HTN, and cold injury model mice. Scale bar, 200 μm. f (left) Representative images of PDGFRβ immunostaining showing pericyte coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex and hippocampus of sham and HTN model mice. (right) Representative images of Aquaporin-4 (AQP4) immunostaining showing astrocyte endfoot coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex and hippocampus of sham and HTN model mice. Scale bar, 200 μm. g Representative images of α-SMA immunostaining showing α-SMA positive cell coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex of sham and HTN model mice. Scale bar, 100 μm. h Quantification of pericyte coverage, astrocyte endfoot coverage, and α-SMA+ cell coverage of CD31+ brain capillaries shown in f and g. (n: the number from 4 mice per group, unpaired two-sided t-test). Data are presented as mean ± SEM. ns, not significant.

Source data

Extended Data Fig. 4 AAV-BI30-mediated transfer facilitated nSMase2 knockdown and reduced EV release in brain endothelial cells.

a Immunoblots showing the downregulation of the nSMase2 (top) and Rab27a (bottom) in Neuro 2a (N2a) cells. β-actin was used as the loading control. b Immunoblots of EV proteins as indicated. Small EVs were collected from equal volumes of supernatants from N2a cells treated with shRNA. β-actin from N2a cells was used as the loading control. The samples shown were obtained from the same experiment and the blots were processed in parallel. c Immunoblots showing the downregulation of the nSMase2 (top) in SVEC4-10 cells and the expression of EV proteins (bottom) as indicated. β-actin in cells was used as the loading control. d Quantification of the nSMase2 in N2a cells. (n = 4 biological replicates, one-way ANOVA with Dunnett’s correction). e Quantification of the protein Rab27a in N2a cells. (n = 4 biological replicates, unpaired two-sided t-test). f Quantification of EV proteins from N2a cells treated with shRNA. (n = 3 biological replicates, one-way ANOVA with Dunnett’s correction). g Quantification of the nSMase2 in SVEC4-10 cells. (n = 3 biological replicates, unpaired two-sided t-test). h Quantification of EV proteins in SVEC4-10 cells. (n = 3 biological replicates, unpaired two-sided t-test). i Representative images showing AAV-BI30-mediated FLAG expression in the brain of mice following intravenous injection. Brain sections from the indicated 8-week-old mice were analyzed 5 weeks after injection. Scale bar, 1 mm. j Representative images showing the inter-organ distribution of AAV-BI30-mediated FLAG expression in mice following intravenous injection. Scale bar, 200 μm. k Specificity of AAV-BI30-mediated transduction of brain endothelial cells in vivo. Images of FLAG expression in the cortex of mice injected with AAV-BI30, co-stained with Lectin, PDGFRβ, GFAP, Iba1, and NeuN. Scale bar, 25 μm. l Immunoblots (left) and quantification (right) of nSMase2 expression in brain microvascular endothelial cells (BMVECs) from mice following intravenous injection. BMVECs were isolated 5 weeks after injection. β-actin from BMVECs was used as the loading control. (n = 4 per group, unpaired two-sided t-test). m-n EV characterization in the CSF 5 weeks following intravenous injection by nano flow cytometry. Representative nano-flow cytometry plots (m) and (n) percentage of endothelial CD31+ small EVs in the total CD63+ small EVs for the indicated groups. (n = 4 per group, unpaired two-sided t-test). Data are presented as mean ± SEM. ns, not significant. Abbreviations: nSMase2, neutral sphingomyelinase 2; shRNA, short hairpin RNA; Scr, a short hairpin RNA targeting scramble sequences; sh nSMase2, short hairpin RNA targeting nSMase2; sh1, a short hairpin RNA targeting nSMase2 with sequence 1; sh2, a short hairpin RNA targeting nSMase2 with sequence 2; sh3, a short hairpin RNA targeting nSMase2 with sequence 3; shRab27a, a short hairpin RNA targeting RAB27A; AAV-sh nSMase2, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting neutral sphingomyelinase 2 (nSMase2); AAV-sh Scr, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting scramble sequences.

Source data

Extended Data Fig. 5 Brain endothelial nSMase2 knockdown had no effects on microvessel structure in the central nervous system.

a (left) Representative images and quantification of PDGFRβ immunostaining showing pericyte coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex and hippocampus of mice treated with AAV-sh Scr or AAV-sh nSMase2. (right) Representative images and quantification of Aquaporin-4 (AQP4) immunostaining showing astrocyte endfoot coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex and hippocampus of mice treated with AAV-sh Scr or AAV-sh nSMase2. Scale bar, 200 μm. b Representative images and quantification of α-SMA immunostaining showing α-SMA positive cell coverage (magenta) of CD31+ brain capillaries (cyan) in the cortex of mice treated with AAV-sh Scr or AAV-sh nSMase2. Scale bar, 100 μm. c Quantification of pericyte coverage, astrocyte endfoot coverage, and α-SMA+ cell coverage of CD31+ brain capillaries shown in a and b. (n: the number from 4 mice per group, unpaired two-sided t-test). Data are presented as mean ± SEM. ns, not significant. Abbreviations: AAV-sh nSMase2, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting neutral sphingomyelinase 2 (nSMase2); AAV-sh Scr, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting scramble sequences.

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Extended Data Fig. 6 Characterization of BEEVs.

a Lactate dehydrogenase (LDH) assay showing comparable cell injury across groups treated with different concentrations of Angiotensin II (AngII). No significant differences were observed among the groups. (n = 6 biological replicates, one-way ANOVA with Tukey’s correction). b Transepithelial electrical resistance (TEER) values over 4 days for brain microvascular endothelial cells (BMVECs) in groups treated with different concentrations of AngII. (n = 4 biological replicates, two-way ANOVA with Tukey’s correction; * AngII 1 μM vs 0 μM, # AngII 10 μM vs 0 μM). c Representative image of the morphology of isolated brain endothelial-derived small extracellular vesicles (BEEVs) visualized by transmission electron microscopy (TEM). Higher-magnification views of the regions indicated by dashed rectangles in the top panel are shown below. Scale bar, top-100 nm; bottom-50nm. d-e Size distribution of isolated BEEVs measured by nanoparticle tracking analysis (NTA, d) and quantification of small EV concentrations (e) from equal volumes of supernatants of BMVECs treated with PBS or AngII. f-g Immunoblots (f) and quantification (g) of extracellular vesicle (EV)-associated proteins as indicated.BEEVs were collected from equal volumes of supernatants of BMVECs treated with PBS or AngII. β-actin from BMVECs was used as the loading control. The samples shown were obtained from the same experiment and the blots were processed in parallel. (n = 4 biological replicates, unpaired two-sided t-test). h Representative images showing the overlap of Rab7-positive vesicles (green) and LAMP1-positive vesicles (amber) in BMVECs as indicated. BMVECs were treated with Vehicle (Veh), 1 μM AngII, 2.5 μM GW4869, or 1 μM AngII + 2.5 μM GW4869. Scale bar, 10 μm. i Pearson’s correlation coefficient (left) and quantification of the percentage of Rab7-LAMP1 colocalization within LAMP1-positive vesicles (right). (n = 3 biological replicates, two-way ANOVA with Tukey’s correction). j LDH assay showing comparable levels of cell injury across groups as indicated. (n = 5 biological replicates, two-way ANOVA with Tukey’s correction). k Immunoblots (left) and quantification (right) of VE-cadherin expression in BMVECs as indicated. (n = 4 biological replicates, two-way ANOVA with Tukey’s correction). l Immunoblots (left) and quantification (right) of EV-associated protein as indicated. BEEVs were collected from equal volumes of supernatants of BMVECs treated with Veh, 1 μM AngII, 1 μM AngII +10 μM Irbesartan, and 1 μM AngII +10 μM Apocynin. (n = 4 biological replicates, one-way ANOVA with Tukey’s correction).Data are presented as mean ± SEM. ns, not significant.

Source data

Extended Data Fig. 7 AngII-treated BEEVs reduced synapse density and excitatory neurotransmitter release in primary neurons in vitro.

a Representative live-cell imaging of PKH-labeled BEEVs in cultured neurons. PKH+ BEEVs (green) were observed within the soma (top) and along neurites (bottom). Scale bar, 2 μm. b Representative images of dendrites from DIV 10 primary neurons in the indicated treatment groups. Neurons were treated with PBS, normal EVs from BMVECs (Ctrl-BEEVs), or AngII-treated EVs from BMVECs (AngII-BEEVs). Dendrites were immunostained for Synaptophysin (SYP) and PSD95. Scale bar, 5 μm. c Quantification of the average density of SYP puncta (left), PSD95 puncta (middle), and SYP/PSD95 co-localized puncta (right) in neurons with the indicated treatments. (n: the number from 4 biological replicates, one-way ANOVA with Tukey’s correction). d Quantification of the relative expression of glutamate (left) and gamma-aminobutyric acid (γ-GABA, right) in the supernatants from neurons with the indicated treatments, 48 hours after treatment. (n: the number from 4 biological replicates, one-way ANOVA with Tukey’s correction). Data are presented as mean ± SEM. Abbreviations: BF, bright field; BEEVs, brain endothelial-derived small extracellular vesicles; BMVECs, brain microvascular endothelial cells; AngII, Angiotensin II; Ctrl, control.

Source data

Extended Data Fig. 8 PEGylation decreased cellular uptake of BEEVs in vitro.

a Immunoblots of GFP and Alix in brain endothelial-derived small extracellular vesicles (BEEVs). Brain microvascular endothelial cells (BMVECs) was transfected with CD63-GFP or GFP plasmid, and BBEVs were collected from equal volumes of conditioned medium. b Immunoblots of PEG and TSG101 in BEEVs after post-insertion with PEG-NHS ester. c-d Immunoblots (c) and quantification (d) of GFP expression in N2a cells. N2a cells were treated with AngII-BEEVs and PEG-modified AngII-BEEVs. β-actin from N2a cells was used as the loading control. (n = 4 biological replicates, one-way ANOVA with Tukey’s correction). Data are presented as mean ± SEM. Abbreviations: AngII, angiotensin II; Ctrl, control; EVs, extracellular vesicles; PEG_EVs, PEGylated extracellular vesicles.

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You, T., Wang, Y., Xu, J. et al. Brain endothelial cell-derived extracellular vesicles (c-BEEVs) as a promising biomarker for brain vascular pathology and cognitive decline. Nat Aging (2026). https://doi.org/10.1038/s43587-026-01117-y

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