Circulating Extracellular Vesicle-Based Biomarkers: Advances, Clinical Implications and Challenges in Coronary Artery Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is a review article on the contribution of extracellular vehicles (EVs) as biomarkers in coronary artery disease (CAD). This article provides a thorough review of the current literature, highlighting promising findings and the methodological challenges that hinder clinical translation. The focus is on circulating EVs and their potential for future clinical applications. The authors advance the idea of mechanistic involvement before designating any molecule as a valid biomarker. The authors discussed several challenges that hinder the integration of EV biomarkers into routine clinical practice, including methodological variability and limited validation, as well as emerging tools that can enhance their potential to improve cost-efficiency and support standardized protocols and clinical feasibility. Overall, this is an engaging, concise, well-written, and informative review, supported by relevant references. I have a few minor recommendations for consideration:
All sections consist of many small paragraphs, and some paragraphs have just 2-3 lines. Authors should combine these paragraphs to create a smooth reading flow and a cohesive story.   

Author Response

We thank the Reviewer for the positive feedback. We are especially grateful, as the comments have substantially improved the quality of our study.

It is a review article on the contribution of extracellular vehicles (EVs) as biomarkers in coronary artery disease (CAD). This article provides a thorough review of the current literature, highlighting promising findings and the methodological challenges that hinder clinical translation. The focus is on circulating EVs and their potential for future clinical applications. The authors advance the idea of mechanistic involvement before designating any molecule as a valid biomarker. The authors discussed several challenges that hinder the integration of EV biomarkers into routine clinical practice, including methodological variability and limited validation, as well as emerging tools that can enhance their potential to improve cost-efficiency and support standardized protocols and clinical feasibility. Overall, this is an engaging, concise, well-written, and informative review, supported by relevant references. I have a few minor recommendations for consideration:
All sections consist of many small paragraphs, and some paragraphs have just 2-3 lines. Authors should combine these paragraphs to create a smooth reading flow and a cohesive story.

We thank the Reviewers for positive comments and revise the Ms accordingly

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The review aims at providing a wide panoramic view of the studies on EVs in the coronary artery disease settings, focusing on their role and potential usefulness in the clinical practice, and includes synoptic tables summarizing the main studies on EVs in the field.

Although riche in several details the manuscript need improvements to reach the publication level and add novel insights to those already available ( e.g., https://doi.org/10.1038/nrcardio.2017.7).

Comments:

• The authors should pay attention in not giving the erroneous clinical definitions :
  10. Heart Disease (coronary heart disease, CHD) and CAD (coronary artery disease) are not superposable (in addition to the numerous websites of cardio foundations and clinics, please see for example https://doi.org/10.1016/j.jacc.2012.02.082).
  11. It is not clear what is the difference between CAD and CCS.
  12. INOCA are not well presented: microvascular angina is a clinical presentation in INOCA due to CMD (DOI: 3390/jcm9092880; https://www.ecrjournal.com/articles/european-society-cardiology-highlights-late-breaking-science-minocaanoca?language_content_entity=en)
• Analogous comment for the EV isolation methods: superiority of combining methodologies is not suggested by ref 26, which compares 5 different methods for obtaining  EVs in 2 types of samples (cell supernatans and plasma)
• Avoid generic claims and be more specific: the mediators and RNAs carried by the EVs may differ in patients with different pathologies which lead to different effects in the patient. It could be interesting to associate EV features with patient phenotype, and the authors should provide. More details, not limiting this important issue to a statement with few references.
• The citations should not be merely presented as “examples” but integrated in a logical pattern reaching paragraph conclusions where possible
• EVs are known carriers of noncoding RNAs, including lncRNAs and miRs. The paragraph “Non-Coding RNAs and CAD: Insights on the State-of-the-Art” is poor of references about strategies.
• Discountinuity in reading is assessed as 2.2 introduced miRs, but 2.3 refers to surface markers as CDs. Specifically, Paragraph 2.2 needs to be carefully reconsidered. It looks like that Lines 150-163 are a sort of preface of paragraph 2.3, whilst lines 144-149 the preface of 3.3.
• AUC or other numerical. Values obatined by other studies are not useful in a review. The authors cited several circ-RNAs and miRs putatively present or found on EVs withouth providing any information about the related patheways / mechanistic role. Because the synopsis. Of the studies is already presented in tabular form, it should be useful trying to explain the role of these RNAs . partially reflecting the EVs function in CAD
• Please check online on Pubmed for bibliography updates (e.g., doi: 10.1016/j.vesic.2025.100070, doi: 10.1186/s13287-025-04390-7.  doi: 10.1186/s40001-025-02649-6, doi: 10.3389/fcvm.2025.1465646.)
• Paragraph 4 should be revised and integrated in paragraphs 5 and 6
• Discussion is too long: please revise and possibly move some observations in the relevant paragraphs
• Proofread the text, paying attention also to typing errors ( e.g. line 110  "wildly" is stated instead of widely).

Author Response

We thank the Reviewer for the positive feedback. We are especially grateful, as the comments have substantially improved the quality of our study.

• The authors should pay attention in not giving the erroneous clinical definitions : Heart Disease (coronary heart disease, CHD) and CAD (coronary artery disease) are not superposable (in addition to the numerous websites of cardio foundations and clinics, please see for example doi.org/10.1016/j.jacc.2012.02.082).

 We have reformulated our wording, citing a new source (line 58-60)

• It is not clear what is the difference between CAD and CCS.

Table CAD (SCAD) and Chronic Coronary Syndrome (CCS) are terms that are often used interchangeably in the literature. However, according to the most recent ESC guidelines, only the term CCS is officially endorsed. We have cited a JACC article that provides a detailed comparison of international guidelines and clarifies the rationale behind this nomenclature change (lines 62 - 65).

• INOCA are not well presented: microvascular angina is a clinical presentation in INOCA due to CMD (DOI: 3390/jcm9092880; https://www.ecrjournal.com/articles/european-society-cardiology-highlights-late-breaking-science-minocaanoca?language_content_entity=en)

We have re-written the paragraph in a more accurate and precise manner (lines 445 - 452)

• Analogous comment for the EV isolation methods: superiority of combining methodologies is not suggested by ref 26, which compares 5 different methods for obtaining EVs in 2 types of samples (cell supernatans and plasma).

Reference 26 has been moved to the previous statement (line 103), as we believe it is more appropriate to cite this article in the context of discussing the different isolation techniques. Additionally, we have reformulated the concept of potentially combining methodologies as a means to enhance the isolation process (lines 103–107).

• Avoid generic claims and be more specific: the mediators and RNAs carried by the EVs may differ in patients with different pathologies which lead to different effects in the patient. It could be interesting to associate EV features with patient phenotype, and the authors should provide. More details, not limiting this important issue to a statement with few references.

The authors have integrated this aspect by introducing a new section titled “2.4 EVs: an additional tool to define disease subtypes.” In this section, we explored EV characteristics in relation to various variables, including demographics (e.g., the EVaging index), comorbidities (such as type 2 diabetes mellitus), and responses to clinical interventions. Furthermore, throughout the review, we have consistently discussed how the effects of ncRNAs can vary depending on the target cell subtype and the patient’s comorbidities.

 

• The citations should not be merely presented as “examples” but integrated in a logical pattern reaching paragraph conclusions where possible

We have integrated these references throughout the review, either by reformulating existing citations or by incorporating additional relevant studies where appropriate. Please see section 3-8.

• EVs are known carriers of noncoding RNAs, including lncRNAs and miRs. The paragraph “Non-Coding RNAs and CAD: Insights on the State-of-the-Art” is poor of references about strategies.

The section previously titled “2.2 Non-Coding RNAs and CAD: Insights on the State-of-the-Art” has been moved to Section 3 and now serves as a concluding paragraph (see lines 290–296), to better align with the overall structure and flow of the manuscript.

The paragraph “2.3 Strategies for Identifying EV RNA Cargo: Computational Analyses vs Functional Approaches” has been renamed to “2.2 Strategies for Identifying EV Cargo: Emerging Role of Computational Analyses.” Additionally, we have included a new reference to support this updated perspective.

• Discontinuity in reading is assessed as 2.2 introduced miRs, but 2.3 refers to surface markers as CDs. Specifically, Paragraph 2.2 needs to be carefully reconsidered. It looks like that Lines 150-163 are a sort of preface of paragraph 2.3, whilst lines 144-149 the preface of 3.3.

Paragraph 2.2, as mentioned above, has been moved to section 3.

• AUC or other numerical. Values obtained by other studies are not useful in a review. The authors cited several circ-RNAs and miRs putatively present or found on EVs withouth providing any information about the related patheways / mechanistic role. Because the synopsis of the studies is already presented in tabular form, it should be useful trying to explain the role of these RNAs, partially reflecting the EVs function in CAD

AUC values have been removed from the main text. Additionally, Table 3, which contained these data, has been repositioned as Supplementary Table 1, as it is not directly pertinent to the main focus of the review.

Initially, we chose not to reiterate mechanistic pathways that have already been extensively covered in previous reviews for each ncRNA included in our work. This decision was made to (1) avoid redundancy and (2) prevent the manuscript from becoming overly lengthy, given the large number of circRNAs and miRNAs discussed. Nevertheless, we agree that highlighting the role of selected ncRNAs can enhance the reader’s understanding of the contribution of EVs to CAD pathophysiology, particularly where conflicting effects have been reported in the literature. For example, see lines 301–337, 349–362, 392–407, and 435–440. Moreover, for several circRNAs, mechanistic insights remain limited in the current literature, which restricted our ability to discuss their functional roles in detail

• Please check online on Pubmed for bibliography updates (e.g., doi: 10.1016/j.vesic.2025.100070, doi: 10.1186/s13287-025-04390-7. doi: 10.1186/s40001-025-02649-6, doi: 10.3389/fcvm.2025.1465646.)

After reviewing the most recent publications, we found it valuable to include the recent work by Limpitikul et al. This has been added as reference 142 in Section 7. We also included a paper from our group which has been recently accepted for publication (Femminò et al. iScience 2025)

• Paragraph 4 should be revised and integrated in paragraphs 5 and 6

As kindly suggested by the Reviewer we integrated the two different sections.

• Discussion is too long: please revise and possibly move some observations in the relevant paragraphs

As kindly suggested by the Reviewer the discussion section has been revised accordingly

• Proofread the text, paying attention also to typing errors ( e.g. line 110  "wildly" is stated instead of widely).

We revised the Ms accordingly