Molecular Mechanisms of Cardiac Fibrosis: A Pathologist’s Perspective

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Several methodological, structural, and presentation issues should be addressed in order to improve the scientific rigor, transparency, and overall quality of the manuscript:

1. A major methodological weakness is the absence of a structured literature search and selection process. The authors do not report the databases searched, search terms, time limits, eligibility criteria, or the number of records screened and included. Moreover, the lack of a PRISMA flow diagram limits transparency and reproducibility, raising concerns regarding potential selection bias and the completeness of the evidence base.
2. The authors should clearly define and justify the inclusion and exclusion criteria to strengthen methodological rigor and allow readers to assess the validity of the literature selection process.
3. The figures lack scale bars and do not provide sufficient methodological explanations or descriptive details, which limits their scientific interpretability and added value.
4. All tables (Tables 1-4) lack explicit bibliographic references. Although the tables summarize multiple mechanisms and associations, the source of each statement is not always clearly traceable. The authors are encouraged to include explicit references within the tables (either per row or in a dedicated column).
5. Several abbreviations used throughout the manuscript are missing from the Abbreviations list. All abbreviations should be clearly defined at first mention and consistently included in the Abbreviations section, in accordance with MDPI formatting requirements.

I hope these comments support the further refinement of the manuscript.

Comments on the Quality of English Language

The English language is generally clear; however, minor grammatical and typographical issues should be corrected.

Author Response

Dear Reviewer 1,

Thank you for your thoughtful and constructive comments, which have significantly improved the methodological clarity, transparency, and overall quality of the manuscript. Below, we address each point raised in detail.

Comment 1: A major methodological weakness is the lack of a structured literature search and selection process. The authors do not report the databases searched, search terms, time limits, eligibility criteria, or the number of records screened and included. Moreover, the lack of a PRISMA flow diagram limits transparency and reproducibility, raising concerns regarding potential selection bias and the completeness of the evidence base.

Response 1: We wish to clarify that this manuscript is intended as a narrative (non-systematic) review of the literature, aiming to integrate morphological and molecular evidence of cardiac fibrosis from an anatomopathological perspective, rather than providing a comprehensive quantitative synthesis of all available evidence. However, to improve methodological transparency, we have added a dedicated section (Materials and Methods, paragraph 2, highlighted in purple) that clearly describes the approach adopted for identifying and selecting the literature, specifying the databases consulted, the main keywords, and the time period considered. Consistent with the narrative nature of the review, a PRISMA diagram, which is specifically recommended for systematic reviews and meta-analyses, has not been included.

Comment 2: The authors should clearly define and justify the inclusion and exclusion criteria to strengthen methodological rigor and allow readers to assess the validity of the literature selection process.

Response 2: Although this is a narrative review, we agree on the importance of clarifying the guiding principles adopted in selecting the literature. To this end, we have explicitly defined the general inclusion and exclusion criteria (Materials and Methods, paragraph 2, highlighted in purple), improving the reader's readability and assessment of the decision-making process.

Comment 3: The figures lack scale bars and do not provide sufficient methodological explanations or descriptive details, which limits their scientific interpretability and added value.

Response 3: We accept the reviewer's suggestion. All figures have been revised to include scale bars, enlargements, and essential methodological details in the legends to improve their scientific interpretability and educational value (highlighted in purple).

Comment 4: All tables (Tables 1–4) lack explicit bibliographic references. Although the tables summarize multiple mechanisms and associations, the source of each statement is not always clearly traceable. Authors are encouraged to include explicit references within the tables (either per row or in a dedicated column).

Response 4: We fully agree with the reviewer. All tables (Tables 1–4) have been revised to include explicit bibliographic references, inserted per row (highlighted in purple), to ensure the traceability of the summarized information.

Comment 5: Several abbreviations used throughout the manuscript are missing from the Abbreviations list. All abbreviations should be clearly defined at first mention and consistently included in the Abbreviations section, in accordance with MDPI formatting requirements.

Response 5: We have conducted a thorough review of the abbreviations used in the manuscript. All abbreviations are now defined at first use and consistently listed in the Abbreviations section (highlighted in purple, before the bibliography), in accordance with MDPI guidelines.

Comment 6: The English language is generally clear; however, minor grammatical and typographical issues should be corrected.

Response 6: The English language has been further revised and grammatical and typographical issues have been corrected.

We again thank Reviewer 1 for his timely and constructive comments.

Sincerely,

The Authors

Reviewer 2 Report

Comments and Suggestions for Authors

This is an important topic for review, and please revise it following suggestions.

• The manuscript does not present a novel conceptual framework, hypothesis, or reinterpretation of cardiac fibrosis. Most sections reiterate canonical profibrotic pathways (TGF-β/SMAD, Wnt/β-catenin, Hippo–YAP/TAZ, and RAAS) that have already been extensively reviewed in the literature. To enhance novelty, the authors should incorporate emerging mediators of cardiac fibrosis and emphasize new mechanistic insights. In particular, recent evidence suggests that Serum Response Factor (SRF) may play an important regulatory role in cardiac fibrosis (PMID: 40164849; PMID: 20498652), and this literature should be critically discussed.

• In addition, mitochondrial dysfunction is increasingly recognized as a key driver of cardiac fibrosis and warrants focused attention. The authors should expand the discussion to include mitochondrial mechanisms and the role of sirtuins in regulating fibrotic remodeling, with reference to recent studies (PMID: 41301698; PMID: 41005588).

• Although the manuscript is presented as a pathologist-centered analysis, histopathology is largely used as illustrative background rather than as an analytical framework. The authors should provide a more comprehensive discussion of available histological and immunohistochemical markers of cardiac fibrosis, including the limitations, sensitivity, specificity, and interpretive challenges of current markers.

• The manuscript should more clearly address how pathological assessment can alter, refine, or contextualize molecular interpretations of fibrosis. Specifically, the authors should discuss how histological findings may support, contradict, or refine conclusions drawn from molecular and omics-based studies.

• Several important unresolved issues in the field are not adequately addressed and should be discussed in greater depth, including:

  • Why antifibrotic therapies have repeatedly failed in clinical trials,

  • Whether fibrosis is a primary causal driver or an epiphenomenon in different cardiac disease contexts,

  • The temporal reversibility of cardiac fibrosis and the extent to which pathology can (or cannot) capture dynamic remodeling processes.

• The manuscript repeatedly implies translational relevance, particularly in relation to sudden cardiac death; however, the supporting evidence remains limited. The authors should expand this section by providing a more critical and evidence-based discussion of how fibrotic patterns contribute to arrhythmogenesis and how pathological findings may inform clinical risk stratification.

• A schematic figure summarizing the key concepts of the review is strongly recommended. This schematic should integrate current knowledge, highlight novel mechanisms discussed in the manuscript, and clearly illustrate the relationship between molecular pathways, histopathological features, and clinical outcomes.

• Finally, the authors must provide detailed information for Figures 1 and 2, including the source of the images (original data versus previously published material), confirmation of permission for reuse if applicable, and full technical details. These should include species, age, and experimental conditions of the samples, as well as clearly labeled scale bars, which are currently missing.

Author Response

Dear Reviewer 2,

We thank Reviewer 2 for their insightful and stimulating comments, which have substantially strengthened the conceptual originality, critical depth, and pathological perspective of the manuscript. We have embraced their suggestions, expanding the discussion on emerging mechanisms, the interpretive role of the disease, and the main unresolved questions in the field.

Comment 1) The manuscript does not present a novel conceptual framework, hypothesis, or reinterpretation of cardiac fibrosis. Most sections reiterate canonical profibrotic pathways (TGF-β/SMAD, Wnt/β-catenin, Hippo–YAP/TAZ, and RAAS) that have already been extensively reviewed in the literature. To enhance novelty, the authors should incorporate emerging mediators of cardiac fibrosis and emphasize new mechanistic insights. In particular, recent evidence suggests that Serum Response Factor (SRF) may play an important regulatory role in cardiac fibrosis (PMID: 40164849; PMID: 20498652), and this literature should be critically discussed.

Response 1: We fully endorse this observation. We have expanded the manuscript by introducing a new subsection dedicated to Serum Response Factor (SRF) as an emerging transcriptional node integrating mechanical, cytoskeletal, and profibrotic signals. SRF is now discussed not as a simple additional pathway, but as a functional bridge between mechanotransduction, fibroblast activation, and extracellular matrix organization, with direct implications for histopathological interpretation. (highlighted in light blue, paragraph 5.5)

The following reference (Miano J. M. (2010). Role of serum response factor in the pathogenesis of disease. Laboratory investigation; a journal of technical methods and pathology, 90(9), 1274–1284. https://doi.org/10.1038/labinvest.2010.104) cannot be inserted because it is too old, and in the materials and methods section, we have specified that the articles used are from 2020 to 2025.

Comment 2) In addition, mitochondrial dysfunction is increasingly recognized as a key driver of cardiac fibrosis and warrants focused attention. The authors should expand the discussion to include mitochondrial mechanisms and the role of sirtuins in regulating fibrotic remodeling, with reference to recent studies (PMID: 41301698; PMID: 41005588).

Answer 2: We have inserted a new subsection on mitochondrial dysfunction, placing particular emphasis on the regulatory role of sirtuins (SIRT1, SIRT3) in controlling fibroblast metabolism, oxidative stress, and tissue stiffness. (highlighted in light blue, paragraph 5.5)

Comment 3) Although the manuscript is presented as a pathologist-centered analysis, histopathology is largely used as an illustrative background rather than an analytical framework. The authors should provide a more comprehensive discussion of available histological and immunohistochemical markers of cardiac fibrosis, including the limitations, sensitivity, specificity, and interpretive challenges of current markers.

Answer 3: We have inserted a new paragraph 8 (highlighted in light blue) that emphasizes the active role of the pathologist as a critical interpreter of molecular data, and the section has been expanded to discuss histological and immunohistochemical markers, including their limitations, sensitivity, specificity, and interpretive challenges.

Comment 4) The manuscript should more clearly address how pathological assessment can alter, refine, or contextualize molecular interpretations of fibrosis. Specifically, the authors should discuss how histological findings may support, contradict, or refine conclusions drawn from molecular and omics-based studies.

Answer 4: We introduced paragraph 9 (highlighted light blue) on the role of pathology as a critical filter of omics data, highlighting the possible discrepancies between molecular activation and structural manifestation.

Comment 5) Several important unresolved issues in the field are not adequately addressed and should be discussed in greater depth, including:

• Why antifibrotic therapies have repeatedly failed in clinical trials,
• Whether fibrosis is a primary causal driver or an epiphenomenon in different cardiac disease contexts,
• The temporal reversibility of cardiac fibrosis and the extent to which pathology can (or cannot) capture dynamic remodeling processes.

Response 5: We thank the Reviewer for this extremely pertinent comment. In accordance with the suggestion, we have substantially expanded the discussion by introducing a new section entitled "Unresolved Questions in Cardiac Fibrosis: Biological and Diagnostic Implications" (paragraph 11, light blue highlighted). In this section, we have systematically addressed the three points raised.

Comment 6) The manuscript repeatedly implies translational relevance, particularly in relation to sudden cardiac death; however, the supporting evidence remains limited. The authors should expand this section by providing a more critical and evidence-based discussion of how fibrotic patterns contribute to arrhythmogenesis and how pathological findings may inform clinical risk stratification.

Response 6: We have expanded paragraph 9 by inserting subsection 9.1. (highlighted light blue) providing a more critical discussion.

Comment 7) A schematic figure summarizing the key concepts of the review is strongly recommended. This schematic should integrate current knowledge, highlight novel mechanisms discussed in the manuscript, and clearly illustrate the relationship between molecular pathways, histopathological features, and clinical outcomes.

Answer 7: We have inserted a new schematic figure (Figure 3) which integrates: canonical and emerging mechanisms (SRF, mitochondria, sirtuins), histopathological characteristics, clinical implications.

Comment 8) Finally, the authors must provide detailed information for Figures 1 and 2, including the source of the images (original data versus previously published material), confirmation of permission for reuse if applicable, and full technical details. These should include the species, age, and experimental conditions of the samples, as well as clearly labeled scale bars, which are currently missing.

Answer 8: Please note that the figures are original to our laboratory. The figures have been revised to include scale bars, enlargements, and essential methodological details in the legends, in order to improve their scientific interpretability and educational value (highlighted in purple).

Best regards,

The Authors

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed the previous comments and have substantially improved the methodological transparency, figure presentation, referencing within tables, and overall clarity of the manuscript. The revisions have strengthened the scientific rigor and readability of the work.

I have no further major concerns.

Author Response

Dear Reviewer1,

Thank you very much and best regards,
The Authors

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly improved the manuscript. I have a few minor suggestions:
In Figures 1 and 2, Reviewer would like to know which species tissue is this? rat/mice/human, age. Please mention this in your figure legend or while explaining these in text. If these figures are original data, add this "Representative images from original experiments conducted by the authors." and any other technical  detail must be added for rigor. 

Insert arrows indicating the concentric accumulation of extracellular matrix and do the same for the MT staining. Similarly, for Figures 2A and 2B, adding arrows would make it easier for the general reader to understand the differences.

Add figure legend for Figure 3 that clearly conveys the main message for this schematic.

Author Response

Dear Reviewer2,

Thank you for your comments and as indicated:

Comment 1: In Figures 1 and 2, Reviewer would like to know which species tissue is this? rat/mice/human, age. Please mention this in your figure legend or while explaining these in text. If these figures are original data, add this "Representative images from original experiments conducted by the authors." and any other technical  detail must be added for rigor. 

Response 1: We have inserted the following sentence in the legend of Figure 1 (highlighted in red): "Perivascular fibrosis in adult human myocardium (47-year-old individual). Representative images of original observations obtained from formalin-fixed, paraffin-embedded left ventricular myocardial tissue collected at autopsy."

We have inserted the following sentence in the legend of Figure 2 (highlighted in red): "Replacement fibrosis in adult human myocardium (63-year-old individual). Representative images of original observations obtained from formalin-fixed, paraffin-embedded left ventricular myocardial tissue collected at autopsy."

We have also inserted the following sentence in Materials and Methods (highlighted in red): "The histological images presented in Figures 1 and 2 derive from archived human myocardial tissue samples collected at autopsy and routinely processed in the Pathology Unit. Tissue samples were fixed in 10% neutral-buffered formalin, paraffin-embedded, sectioned at 3–4 µm thickness, and stained with Hematoxylin-Eosin and Masson's trichrome according to standard diagnostic protocols."

Comment 2: Insert arrows indicating the concentric accumulation of extracellular matrix and do the same for the MT staining. Similarly, for Figures 2A and 2B, adding arrows would make it easier for the general reader to understand the differences.

Response 2: We have inserted arrows in Figures 1 and 2 and in the legends (highlighted in red).

Comment 3: Add figure legend for Figure 3 that clearly conveys the main message for this schematic.

Response 3: The legend of Figure 3 has been rewritten to clearly convey the central message of the schematic illustration and its integrative significance.

Thank you and kind regards,

The Authors