Differences in Plasma Extracellular Vesicles of Different Origin in On-Pump Versus Off-Pump Cardiac Surgery

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the study, the authors established that platelet- and erythrocyte-derived CD41+CD235a+ EVs were found to be significantly decreased after on-pump surgery, whereas T-regulatory cell-derived EVs increased after on-pump procedures. The findings are impressive and practically useful. However, I would like to make some suggestion regarding the findings interpretation.

1. The authors might indicate whether they used FMO control for flow cytometry rpocedure.

2. 7-lead electrocardiography is recommended for arhythmia detected, but not for ST segment changes evaluation. Please, check and correct.

3. It remaines unclear what underlying mechanisms were involved in the EV secretion from different cells.

Author Response

We appreciate reviewer for the attention to the manuscript as well as for the remarks and questions. We analyzed all of them and prepared the answers.

1. The authors might indicate whether they used FMO control for flow cytometry rpocedure.

We appreciate reviewer for the valuable remark. We agree that use of FMO controls increase the accuracy of measurement in flow cytometry. Besides need for these controls is mentioned in “MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments” (https://doi.org/10.1080/20013078.2020.1713526). Thus, we have added information in “Extracellular vesicles” subsection of the Materials and methods section.

 

Addition in “Extracellular vesicles”:

Single-stained controls were performed to identify whether each fluorochrome in the panel affecting measurements of another fluorescent reagents as well as for compensation validation…. All isotype controls and single-stained controls were stained and measured in the same conditions and concentrations as the appropriate fluorochrome-marked antibodies.

 

Meanwhile, we did not perform fluorescence minus one (FMO) controls for immunophenotyping the main T and B cell populations. Since we focused on linear markers like CD3, CD4, CD8, CD19, and CD45, additional FMO controls were not necessary for our study design.

 

2. 7-lead electrocardiography is recommended for arhythmia detected, but not for ST segment changes evaluation. Please, check and correct.

We understand the reviewer's concerns. Meanwhile, we use modern guidelines and trends in medicine. In particular, in «Kaplan’s essentials of cardiac anesthesia for cardiac surgery» monitoring of a patient during CABG is described as follows: “On arrival in the operating room, the patient undergoing CABG should have routine monitors placed, including pulse oximetry, noninvasive BP, and the ECG. A five-lead system is standard for patients undergoing cardiac surgery. Monitoring leads V5 and II allows detection of 90% of ischemic episodes and assessment of the rhythm to diagnose various atrial and ventricular arrhythmias” (Kaplan JA. Kaplan’s Essentials of Cardiac Anesthesia [Internet]. Kaplan’s Essentials of Cardiac Anesthesia. Elsevier; 2018. 1–745 p. https://linkinghub.elsevier.com/retrieve/pii/C20120061510).

Besides, Kouz K. et al. stated that “today, intraoperative heart rhythm and rate monitoring with electrocardiography is mandated by the European recommendations for standards of monitoring during anesthesia and recovery (Klein AA, Meek T, Allcock E, Cook TM, Mincher N, Morris C, Nimmo AF, Pandit JJ, Pawa A, Rodney G, Sheraton T, Young P. Recommendations for standards of monitoring during anaesthesia and recovery 2021: Guideline from the Association of Anaesthetists. Anaesthesia. 2021;76:1212–23. doi: 10.1111/anae.15501.) and the American Society of Anesthesiologists Standards for Basic Anesthetic Monitoring (Committee on Standards and Practice Parameters. (2020) Standards for Basic Anesthetic Monitoring of the American Society of Anesthesiologists. Available online: https://www.asahq.org/standards-and-practice-parameters/standards-for-basic-anesthetic-monitoring). In the operating room, electrocardiography systems with three and five electrodes are most commonly used. The limb leads are typically placed on the shoulders, and the placement of the single precordial lead is variable and depends on the surgical procedure. The V5 lead is most sensitive to ST-segment changes, capturing 75% of events; V4 captures about 60% of ST-segment changes; the other precordial locations are significantly less sensitive (London MJ, Hollenberg M, Wong MG, Levenson L, Tubau JF, Browner W, Mangano DT. Intraoperative myocardial ischemia: localization by continuous 12-lead electrocardiography. Anesthesiology. 1988;69:232–41. doi: 10.1097/00000542-198808000-00013. )”.

Thus, in our clinics we use a cable with 5 electrodes and 5-lead or 7-lead ECG system. During heart surgery, we typically use only one precordial electrode, which is V5 and we suppose that enough for arrhythmia detection and for ST segment changes evaluation.

3. It remaines unclear what underlying mechanisms were involved in the EV secretion from different cells.

We agree with the reviewer that identifying the origin of EVs is one of the most interesting and challenging questions. At this stage, our aim was to identify differences in concentrations of EVs of different origins during the postoperative period in patients undergoing off-pump and on-pump surgery. Our ability to identify the origin of EVs based on the presence of receptors in their structure opens up exciting possibilities for future investigations. We hope to further investigate the mechanisms of EV production by different types of cells.

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This is a review of the manuscript “Differences in plasma extracellular vesicles of different origin on-pump versus off-pump cardiac surgery” by Aquino et al.

In regard to the manuscript, I believe it is interesting but I have some concerns. Please see my comments below. I propose minor revisions.

Abstract

Abstract is ok but fails to address actual achievements of this study. To mentioned that numerous correlations were found is too vague as, I’m certain, you agree

Keywords

The last keyword is almost a keyphrase, too long

Introduction

I appreciate a short and direct conclusion; however, this one fails at fully stating the goals of the study. The last paragraph must always clearly state the overall goals of the study and address how they are expected to impact both academic and medical fields. Please improve it

Methods

Numbers > 9 are written in numeric form, so, thirty six should be 36.

Comorbidities are medical conditions that coexist alongside a primary diagnosis, in this way, I’m not sure if age is a comorbidity

Methods are well described

Results are well described and are interesting, however, it is hard to assess their real meaning since the discussion is done separately. I would suggest the merging of results and discussion sections into one and that each set of results was directly and independently discussed. This would improve the clarity of the document and would allow to divide the huge discussions section into portions much easier to read

Conclusions fail to address the real impact of the achieved results in the academic and medical fields. The work has merit but it must also have a point, an impact in real life

35% of repetition rate was detected by Turnitin. This must be fixed.

 

 

Author Response

We appreciate reviewer for the attention to the manuscript as well as for the remarks and questions. We analyzed all of them and prepared the answers. Besides we performed some corrections in almost all sections in the manuscript. We hope that revised version of the paper is much better and we appreciate reviewer for this. 

1. Abstract

Abstract is ok but fails to address actual achievements of this study. To mentioned that numerous correlations were found is too vague as, I’m certain, you agree

We are agreeing with the reviewer. Thus, we performed corrections and additions in the text of the Abstract. We think that now it is clearer, more concrete, and easier to read.

 

Revised Abstract

Coronary artery bypass grafting (CABG) using cardiopulmonary bypass (CPB) causes a systemic inflammatory response that can worsen patient outcomes. Off-pump surgery has been associated with a reduced inflammatory response. The precise mechanisms and the role of extracellular vesicles (EVs) in this context are not fully understood. This study aimed to investigate the early immune response, including main T- and B-lymphocyte subsets, cytokine profiles, and plasma EVs, in patients undergoing off-pump (n=18) and on-pump (n=18) CABG. Thirty-six patients undergoing isolated CABG were enrolled in this randomized control study. Pre- and 24-hours post-operative blood samples were analyzed for immune cell populations, cytokine levels, and plasma EV phenotyping. Off-pump CABG triggered a milder immune response than on-pump surgery. On-pump surgery led to greater changes in circulating EVs, particularly platelet- (CD62P+), endothelial- (CD31+), and B-cell-derived (CD19+) objects, as well as platelet- and erythrocyte-derived aggregates (CD41+CD235a+). Levels of platelet-derived EVs, expressing both constitutional and activation markers (CD41+CD62P+) decreased in both groups of patients 24 hours after surgery. On-pump cardiac procedures led to an increase in T-regulatory cell-derived EVs (CD73+CD39+), suggesting a potential mechanism for immune suppression compared to off-pump surgery. There were numerous correlations between EV levels and cytokine profiles following on-pump surgery, hinting at a close relationship. Leucocyte-derived EVs exhibited positive correlations with each other and with GRO, but showed negative correlations with endothelial-derived EVs (CD90+ and CD31+). Additionally, CD73+ EVs demonstrated positive correlations with platelet counts and with erythrocyte-derived CD235a+ EVs. EV changes were significantly greater after on-pump surgery, highlighting a more pronounced response to this type of surgery and emphasizing the role of EVs as regulators of post-surgical inflammation.

 

 

2. Keywords

The last keyword is almost a keyphrase, too long

Keywords were corrected.

3. Introduction

I appreciate a short and direct conclusion; however, this one fails at fully stating the goals of the study. The last paragraph must always clearly state the overall goals of the study and address how they are expected to impact both academic and medical fields. Please improve it

We agree that the introduction section was not finalized in its current form. To address this, we have added a paragraph at the end of the section and revised the aim statement.  

Addition in the text

Thus, the majority of research comparing off-pump and on-pump open heart surgery has focused on measuring levels of cytokines [20], number of studies focused on cellular response of the immune system [21], basically investigating only main T- and B-cell subsets, and only few manuscripts dedicated to participation of EVs. Meanwhile, understanding the fine-tuning mechanisms of immune response regulation seems to be a key factor in improving the quality of open-heart surgery. The present study is aimed to comprehensively explore early immune response, including dynamics of T- and B-lymphocyte subsets, various cytokines and plasma extracellular vesicles, on coronary artery bypass grafting surgery performed in off-pump or on-pump conditions.

4. Methods

Numbers > 9 are written in numeric form, so, thirty six should be 36.

The number of patients is corrected to “36”

5. Comorbidities are medical conditions that coexist alongside a primary diagnosis, in this way, I’m not sure if age is a comorbidity

We apologize for this mistake. The table 1 was changed. The name of the column was changed from the “comorbidities” to “parameters”.

Methods are well described

6. Results are well described and are interesting, however, it is hard to assess their real meaning since the discussion is done separately. I would suggest the merging of results and discussion sections into one and that each set of results was directly and independently discussed. This would improve the clarity of the document and would allow to divide the huge discussions section into portions much easier to read

We carefully considered the reviewer's suggestion to merge the results and discussion sections, weighing the pros and cons of this approach. Ultimately, we decided that our current manuscript structure best presents our findings. We apologize for any disagreement.

7. Conclusions fail to address the real impact of the achieved results in the academic and medical fields. The work has merit but it must also have a point, an impact in real life

 We agree with the reviewer.  We have expanded the conclusion section with more specific information and our perspective on the implications of the results.

Addition in the conclusion section

Besides, on-pump surgery led to greater changes in circulating EVs, particularly platelet- (CD62P+), endothelial- (CD31+), and B-cell-derived (CD19+). There were numerous correlations between EV levels and cytokine profiles following on-pump surgery, hinting at a close relationship. Leucocyte-derived EVs exhibited positive correlations with each other and with GRO, but showed negative correlations with endothelial-derived EVs (CD90+ and CD31+). Additionally, CD73+ EVs demonstrated positive correlations with platelet counts and with erythrocyte-derived CD235a+ EVs. EV changes were significantly greater after on-pump surgery, highlighting a more pronounced response to this type of surgery and emphasizing the role of EVs as regulators of post-surgical inflammation. This study revealed patterns in the early postoperative immune response, highlighting the crucial role of extracellular vesicles in its regulation. Understanding the fine-tuning mechanisms of this immune response holds significant promise for improving open-heart surgery outcomes.

8. 35% of repetition rate was detected by Turnitin. This must be fixed.

 We agree with the reviewer. The originality report indicates a 35% similarity index. However, most of this similarity stems from repeated use of material and methods sections from our own previous publications, detailing our methodology approaches. These sections are all properly referenced, as are all repeated sections in the discussion.