Journal of Cellular and Molecular Medicine
Abdominal aortic aneurysm (AAA) is the most prevalent and lethal form of arterial aneurysm, frequently manifesting asymptomatically until a catastrophic rupture occurs. While various diagnostic imaging tools and several potential biomarkers have been explored for the purpose of early AAA screening, the usage of liquid biopsy such as extracellular vesicles (EVs)‐carried protein for the early diagnosis of AAA is still being overlooked. In this study, we enrolled 18 AAA patients and nine healthy normal controls, including data from the National Drug Clinical Trial Organisation—Vascular Surgery (NDCTO) (in‐house cohort) and the Second Clinical Medical College, Jinan University (Shenzhen People's Hospital) (external cohort). We employed Olink's proximity extension assay (PEA) technology based on the plasma EV proteins and first comprehensively characterised the proteomics landscape in circulating EV underlying AAA disease development. A complex profile of differential EV proteins and EV protein–protein interactions network in AAA patients was identified. The differentially expressed EV proteins in AAA patients were found to be significantly associated with several enriched pathways, including the cellular response to cytokine stimuli, inflammatory response, and the regulation of the glucocorticoid receptor (GR) pathway. Moreover, five hub proteins were identified as being of particular significance: these were Interleukin‐4, Interleukin‐6, MCP‐1, Neurturin, and Oncostatin‐M. The Olink proteomics technique was utilised in order to identify these proteins. The significance of these proteins was further validated through Western blotting and enzyme‐linked immunosorbent assay (ELISA) in the external cohort. The five EV proteins displayed reliable performance and robustness for distinguishing AAA from healthy people, revealing high accuracy with AUC values of 0.760, 0.840, 0.800, 0.840, and 0.900, respectively. The present study has revealed the plasma EV proteins landscape within AAA and further uncovered their potential roles in the pathogenesis of the disease. This presents a new direction for clinical diagnosis and management of AAA. Consequently, these five EV proteins have the potential to serve as useful biomarkers for the diagnosis and prediction of AAA. Further research is warranted to explore their potential as therapeutic targets.
Chaoyang Yu, Ge Zhang, Shaotong Pei, Yifei Zhang, Peiyu Yuan, Renying Miao, Kaisaierjiang Kadier, Pengpeng Zhang, Tianshu Gu, Ruhao Wu, Haonan Zhang, Shiqian Zhang, Bo Yang, Han Wu, Yudi Xu, Ke Hu, Qingfei Xu, Yaxin Chen, Jinliang Wang, Zongao Cai, Junnan Tang, Teng Li, Yan Song. Deciphering the Proteomic Landscape of Circulating Extracellular Vesicles in Human Abdominal Aortic Aneurysm. Journal of Cellular and Molecular Medicine. 2025;29(15):e70725. https://doi.org/10.1111/jcmm.70725
Deciphering the Proteomic Landscape of Circulating Extracellular Vesicles in Human Abdominal Aortic Aneurysm
A two-center exploratory study profiled 92 circulating EV-associated proteins in AAA and evaluated five candidates by Western blot and ELISA. External sample size, assay-dependent expression differences and wide ROC uncertainty are retained explicitly.
The reported enrollment is consistent with (10+8) AAA patients and (5+4) controls. Protein measurements and repeated assays are not new participants. No independence from participants in other publications is inferred. External validation means assay and ROC assessment in 8 cases and 4 controls, not a prospective screening study. Specimen and isolation descriptions differ between source sections.
Can targeted circulating extracellular-vesicle protein profiling identify AAA-associated candidates that merit external assay evaluation, and how consistent are their signals across Western blot, ELISA and ROC analyses?
This two-center human study enrolled 18 AAA patients and 9 healthy controls: 10 cases and 5 controls in the in-house cohort, and 8 cases and 4 controls in the external cohort. A targeted Olink proximity-extension workflow assessed 92 EV-associated proteins and prioritized five candidates. External Western blot and ELISA did not show uniformly concordant findings: MCP-1 direction differed between the two reported analyses, and Neurturin was not significant by Western blot but lower by ELISA. The article reports single-marker ROC AUCs from 0.760 to 0.900 in the small external comparison, with wide confidence intervals. Different passages also describe venous plasma, serum EVs and intraoperative aortic blood; those specimen descriptions are not silently treated as one validated routine blood-screening protocol. The evidence is exploratory biomarker association and external assay assessment, not prospective AAA detection, rupture prediction or a therapeutic target validation.
Two-center human case-control evidence; no longitudinal prediction of incident AAA or rupture.
| Measure | Value |
|---|---|
| In-house AAA / controls | 10 / 5 |
| External AAA / controls | 8 / 4 |
| Article-reported total AAA / controls | 18 / 9 |
Source locator: Abstract; Methods 2.1; Results 3.4; Fig. 6
The source describes venous plasma, serum EVs and intraoperative aortic blood in different sections; a single standardized peripheral-blood screening workflow is not assumed.
| Measure | Value |
|---|---|
| AAA vesicle median diameter | 85.18 nm |
| Marker findings | TSG101, ALIX and CD9 detected; Calnexin not detected |
| Isolation reporting | ExoQuick in Methods 2.3; ultracentrifugation in the external-validation Methods 2.9 |
Source locator: Methods 2.2-2.7 and 2.9; Results 3.1; Figs. 1-2
Olink targeted-panel measurements are not an unbiased whole proteome or direct evidence of a causal protein pathway.
| Measure | Value |
|---|---|
| Analyzed protein panel | 92 proteins |
| Reported differential set | 5 proteins: 3 upregulated and 2 downregulated |
| Candidate names | IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M |
Source locator: Methods 2.7-2.8; Results 3.2-3.4; Figs. 3-4
The evidence is reported without inventing a reconciled expression direction or a technical explanation for the discrepancy. The ELISA prose uses the term tumour suppressor-M; Oncostatin-M naming here follows the five-analyte list and Fig. 6 labels, not an additional measured protein.
| Measure | Value |
|---|---|
| External sample | 8 AAA patients; 4 healthy controls |
| Western blot | IL-4, IL-6, MCP-1 and Oncostatin-M higher; Neurturin not statistically significant |
| ELISA | IL-4, IL-6 and Oncostatin-M higher; MCP-1 and Neurturin lower |
Source locator: Results 3.4; Fig. 6A-C; Discussion
AUCs are mapped in the explicit candidate order in the Abstract/Results; they are not a five-protein combined model or prospective diagnostic performance.
| Measure | Value |
|---|---|
| IL-4 AUC | 0.760 |
| IL-6 AUC | 0.840 |
| MCP-1 AUC | 0.800 |
| Neurturin AUC | 0.840 |
| Oncostatin-M AUC | 0.900 |
| Precision qualification | Wide confidence intervals; the reported interval for AUC 0.760 is 0.486-1.000 |
Source locator: Abstract; Results 3.4; Fig. 6D
These associations annotate the observed protein set and do not demonstrate experimentally manipulated pathways.
Source locator: Methods 2.8; Results 3.3; Fig. 5
This two-center study enrolled 18 AAA patients and 9 controls across discovery and external-assay phases.
Evidence: KF1
Characterization supports the vesicle preparation, but source sections differ in blood compartment and isolation descriptions.
Evidence: KF2
A 92-protein panel prioritized IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M.
Evidence: KF3
No: MCP-1 direction and Neurturin findings differed, so the study provides qualified cross-assay evidence.
Evidence: KF4
Single-marker AUCs range from 0.760 to 0.900 in a small external case-control comparison; no combined model or prospective screening benefit is established.
Evidence: KF5
Enrichment and network analyses implicated cytokine, chemokine, AP-1 and glucocorticoid-receptor contexts as computational annotations.
Evidence: KF6
| Row ID | Component | Context | Finding | Evidence Level | Scope | Source Locator | Evidence refs |
|---|---|---|---|---|---|---|---|
| evidence-aaa-ev-cohorts | Two-center human design | In-house and external case-control samples | The study combined a 15-person in-house comparison with a 12-person external comparison. In-house AAA / controls: 10 / 5; External AAA / controls: 8 / 4; Article-reported total AAA / controls: 18 / 9 | Human observational study | No incident-disease or rupture prediction. | Abstract; Methods 2.1; Results 3.4; Fig. 6 | KF1 |
| evidence-aaa-ev-specimen | Vesicle and specimen characterization | Circulating EV-associated fraction | Circulating vesicle preparations were characterized by particle morphology, size and protein markers. AAA vesicle median diameter: 85.18 nm; Marker findings: TSG101, ALIX and CD9 detected; Calnexin not detected; Isolation reporting: ExoQuick in Methods 2.3; ultracentrifugation in the external-validation Methods 2.9 | Experimental specimen characterization | Not a harmonized routine peripheral-blood screening protocol. | Methods 2.2-2.7 and 2.9; Results 3.1; Figs. 1-2 | KF2 |
| evidence-aaa-ev-protein-screen | Targeted candidate discovery | 92 Olink protein measurements | A targeted 92-protein profiling workflow prioritized five differentially expressed candidate proteins. Analyzed protein panel: 92 proteins; Reported differential set: 5 proteins: 3 upregulated and 2 downregulated; Candidate names: IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M | Targeted human protein association | Not unbiased proteomics or a causal pathway experiment. | Methods 2.7-2.8; Results 3.2-3.4; Figs. 3-4 | KF3 |
| evidence-aaa-ev-assay-comparison | Cross-assay external evaluation | 8 AAA cases and 4 controls; Oncostatin-M naming follows the candidate list and Fig. 6 labels. | External Western blot and ELISA findings were assay-dependent rather than uniformly concordant for all five candidates. External sample: 8 AAA patients; 4 healthy controls; Western blot: IL-4, IL-6, MCP-1 and Oncostatin-M higher; Neurturin not statistically significant; ELISA: IL-4, IL-6 and Oncostatin-M higher; MCP-1 and Neurturin lower | External assay evidence with discordance | Do not claim uniform validation of all five candidates. | Results 3.4; Fig. 6A-C; Discussion | KF4 |
| evidence-aaa-ev-roc | Single-marker ROC evidence | Small external case-control comparison | The article reports single-protein ROC discrimination in the small external comparison. IL-4 AUC: 0.760; IL-6 AUC: 0.840; MCP-1 AUC: 0.800; Neurturin AUC: 0.840; Oncostatin-M AUC: 0.900; Precision qualification: Wide confidence intervals; the reported interval for AUC 0.760 is 0.486-1.000 | Exploratory diagnostic discrimination | No combined panel model, prospective validation or rupture prediction. | Abstract; Results 3.4; Fig. 6D | KF5 |
| evidence-aaa-ev-pathways | Protein-set functional interpretation | Predicted network/enrichment analysis | Enrichment and network analyses associated the protein candidates with inflammatory and signaling contexts. Reported contexts: Cellular response to cytokines, inflammatory response, chemokine signaling, AP-1 and glucocorticoid-receptor pathways | Computational enrichment | Annotations are not validated therapeutic targets. | Methods 2.8; Results 3.3; Fig. 5 | KF6 |
The article reports 18 AAA patients and 9 controls: 10/5 in-house and 8/4 external. These numbers describe the two reported cohorts, not additional participants per assay.
Evidence: KF1
No. The study examined an isolated circulating vesicle-associated fraction with a targeted 92-protein Olink panel. Specimen wording also differs between source sections.
Evidence: KF2, KF3
IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M. They are candidate biomarkers in the analyzed samples, not approved diagnostic markers.
Evidence: KF3
No. Western blot and ELISA differed for MCP-1 direction and Neurturin significance/direction. The evidence should be cited as assay-dependent external evaluation, not uniformly concordant validation.
Evidence: KF4
In the article-listed order IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M, AUCs were 0.760, 0.840, 0.800, 0.840 and 0.900. The external group was small and confidence intervals were wide. These are single-marker comparisons, not a combined five-protein model.
Evidence: KF5
No. It reports association, small external assay/ROC comparisons and computational annotations, not prospective rupture prediction or therapeutic target validation.
Evidence: KF4, KF5, KF6
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