The Emerging Role of Endothelial Ion Channels in the Control of Human Microcirculation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a strong and thoughtfully assembled review and the depth of mechanistic discussion is a clear strength. The manuscript provides a valuable synthesis of endothelial ion channel biology and its relevance to microvascular control. The comments below are intended to help improve clarity, balance, and translational focus, particularly with respect to the distinction between human and animal evidence. 

1. Human evidence versus animal extrapolation:  Although the Abstract and Introduction emphasize human microcirculation (pp. 1–2), a substantial portion of Section 2 (Sections 2.1–2.5, pp. 3–11) relies primarily on animal studies. In particular, the EDH mechanisms discussed in Sections 2.2 and 2.3 (pp. 5–9), including SKCa/IKCa activation and downstream involvement of KIR2.1 and KATP channels, are largely derived from rodent models, without always clearly indicating which elements have been functionally demonstrated in human microvessels. More explicit labeling of animal-derived findings within these sections, along with brief notes where direct human evidence is limited or absent, would improve clarity and better align the manuscript with its stated focus.

2. Section 2 (pp. 3–11) is technically strong but considerably longer than Section 3, which is presented as the main human-focused part of the review. Some mechanistic descriptions appear repeatedly. For example, InsP₃R–STIM–Orai signaling is introduced in Section 2.1 (pp. 3–4) and revisited with similar explanatory detail in Sections 2.4–2.5 (pp. 8–9). Likewise, EDH signal propagation via SKCa/IKCa and downstream potassium channels is described in detail across Sections 2.2 and 2.3 (pp. 5–8), with overlapping conceptual framing.Streamlining these repeated sections would create space to expand Section 3 with a deeper discussion of human in vivo methodologies, inter-individual variability, and experimental limitations, thereby strengthening the translational emphasis of the review.

3. In Section 3.1.3 (pp. 13–14), the role of EDH in cutaneous active vasodilation is inferred mainly from pharmacological inhibition studies using TEA, BaCl₂, and L-NAME. Given the limited specificity of TEA, conclusions regarding SKCa/IKCa involvement should be framed more cautiously. The proposal that KATP channels act as a “backup vasorelaxant pathway” is intriguing but currently speculative; explicitly identifying this as a working hypothesis and clarifying whether direct molecular or electrophysiological evidence exists in human dermal microvascular endothelial cells would strengthen this section.

4. In Sections 3.1.2 and 3.1.4 (pp. 12–15), TRPV1, TRPV3, and TRPA1 are discussed in relation to cutaneous vasodilation, but the cellular source of these effects is not always clearly defined. In several cases, endothelial involvement is implied, where the cited evidence instead points to sensory neurons or keratinocytes. More clearly distinguishing endothelial versus non-endothelial mechanisms, particularly when interpreting skin blood flow data, would improve clarity and avoid overinterpretation.

5. Piezo1 is described as mediating both vasodilation and vasoconstriction through different mechanisms, but this dual role is not always clearly presented. Organizing the discussion by shear stress conditions and clearly indicating which findings are based on human versus animal studies would improve clarity.

Minor points

1. There are instances of inconsistent terminology and capitalization that should be corrected, such as “Edh” versus “EDH” (pp. 5–7) and “Skca/ikca” versus “SKCa/IKCa” (pp. 8–9).
2. Minor inconsistencies in terminology and capitalization are present in Section 2. For example, EDH is variably written as “Edh” and “EDH” on pp. 5–7, and SKCa/IKCa channels appear with inconsistent capitalization (e.g., “Skca/ikca” versus “SKCa/IKCa”) on pp. 8–9

Overall assessment: This is a high-quality and authoritative review that makes a valuable contribution to the field. Addressing the minor points raised above will further improve clarity and strengthen the manuscript.

Author Response

Please, see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

- 

A significant amount of the text is based on animal research, particularly mouse and rat models, despite the declared focus being on human microcirculation. Although this makes sense given the scarcity of human data, the difference between

direct proof from humans,

ex vivo research on human tissues, and

Extrapolations from animal models ought to be made clearer throughout the text.

Conclusion:

Conclusions drawn from animal research should be clearly labeled, and their limits when applied to people should be emphasized.

Include brief summary tables that show which ion channels have direct evidence in humans as opposed to roles that are inferred or indirect.

 

- The sections on Ca2+ signaling, TRP channels, and EDH are very technical and comprehensive. Although suitable for experts, this could make it less accessible to a larger IJMS readership.

Conclusion:

Repeated explanations of the InsP₃–Ca²⁺–SKCa/IKCa connection are examples of condensed mechanistic repetition.

Transfer extremely precise molecular descriptions to additional material or graphics.

 

- Differentiating Between Endothelial and Non-Endothelial Effects

Vasodilatory effects are not always evident in a number of sections (such as TRPV1, TRPA1, and TRPV3).

endothelium-derived,

Perivascular nerves mediate this

or secondary to smooth muscle or keratinocytes.

Advice:

Include clear remarks that differentiate endothelial localization from parenchymal or neuronal effects, especially in the circulation of the skin.

 

• The following papers can be added to the current research:
• 1: Jackson, W. F. (2022). Endothelial Ion Channels and Cell-Cell Communication in the Microcirculation. Frontiers in Physiology, 13. https://doi.org/10.3389/fphys.2022.805149
• 2: Nave, Op. (2020). Modification of Semi-Analytical Method Applied System of ODE. Modern Applied Science, 14(6), 75. https://doi.org/10.5539/mas.v14n6p75    

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please, see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review systematically summarizes the role of endothelial ion channels in the regulation of human microcirculation. It is detailed, clearly structured, and of significant physiological and pathological importance. However, there is considerable room for improvement in the integration of human-specific evidence, in-depth exploration of clinical translational potential, and optimization of diagrams and structures.

1. The article mentions endothelial ion channels as "drugatable targets," but does not systematically summarize current drug development progress or preclinical/clinical trial evidence.

2. The existing diagrams are few, and some mechanism descriptions rely on text, which is not conducive to readers' understanding of complex signaling pathways. It is recommended to add a "Signal Integration Network Diagram of Endothelial Ion Channels in Human Microcirculation," integrating pathways such as Ca²⁺, K⁺, NO, and EDH. A "Comparison Table of Endothelial Channel Expression and Function in Different Vascular Beds (Skin, Muscle, Brain)" should be added. More molecular mechanism annotations should be added to Figure 5 (Skin Blood Flow Regulation), such as the specific action sites of TRPV1/TRPV4 in heat stress.

3. Add a "Future Research Directions" paragraph before the "Conclusion" section, proposing the development of endothelial-specific channel modulators, the use of multi-omics technologies to map human microcirculatory endothelial channels, and the establishment of a human microvascular-on-a-chip model for drug screening.

4. The discussion of "how endothelial ion channel dysregulation specifically leads to disease" is too general. It is recommended to combine clinical research to explain the expression/functional changes of specific channels (such as TRPV4 and Kir2.1) in hypertension, diabetes, and Alzheimer's disease.

5. The article mentions endothelial ion channels as "drugatable targets," but does not systematically summarize existing drug development progress or preclinical/clinical trial evidence. It is recommended to add a "Therapeutic Potential and Drug Development" section, summarizing known agonists/antagonists targeting TRPV1, TRPV4, Piezo1, etc., and their experimental progress in cardiovascular/neurological diseases.

Comments on the Quality of English Language

This review systematically summarizes the role of endothelial ion channels in the regulation of human microcirculation. It is detailed, clearly structured, and of significant physiological and pathological importance. However, there is considerable room for improvement in the integration of human-specific evidence, in-depth exploration of clinical translational potential, and optimization of diagrams and structures.

1. The article mentions endothelial ion channels as "drugatable targets," but does not systematically summarize current drug development progress or preclinical/clinical trial evidence.

2. The existing diagrams are few, and some mechanism descriptions rely on text, which is not conducive to readers' understanding of complex signaling pathways. It is recommended to add a "Signal Integration Network Diagram of Endothelial Ion Channels in Human Microcirculation," integrating pathways such as Ca²⁺, K⁺, NO, and EDH. A "Comparison Table of Endothelial Channel Expression and Function in Different Vascular Beds (Skin, Muscle, Brain)" should be added. More molecular mechanism annotations should be added to Figure 5 (Skin Blood Flow Regulation), such as the specific action sites of TRPV1/TRPV4 in heat stress.

3. Add a "Future Research Directions" paragraph before the "Conclusion" section, proposing the development of endothelial-specific channel modulators, the use of multi-omics technologies to map human microcirculatory endothelial channels, and the establishment of a human microvascular-on-a-chip model for drug screening.

4. The discussion of "how endothelial ion channel dysregulation specifically leads to disease" is too general. It is recommended to combine clinical research to explain the expression/functional changes of specific channels (such as TRPV4 and Kir2.1) in hypertension, diabetes, and Alzheimer's disease.

5. The article mentions endothelial ion channels as "drugatable targets," but does not systematically summarize existing drug development progress or preclinical/clinical trial evidence. It is recommended to add a "Therapeutic Potential and Drug Development" section, summarizing known agonists/antagonists targeting TRPV1, TRPV4, Piezo1, etc., and their experimental progress in cardiovascular/neurological diseases.

Author Response

Please, see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors revised the paper  but need a professional English editor

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Dear Reviewer 2, the manuscript has been reviewer by a professional English editor.

Kind regards,

Francesco

 

Reviewer 3 Report

Comments and Suggestions for Authors

The author has addressed the reviewers' comments and revised the manuscript accordingly, resulting in marked improvements compared to the original version. I have no further suggestions at this stage.

Comments on the Quality of English Language

The author has addressed the reviewers' comments and revised the manuscript accordingly, resulting in marked improvements compared to the original version. I have no further suggestions at this stage.

 

Author Response

Dear Reviewer 3,

We do thank you for your valuable efforts to improve our work. We are really grateful!

With my kind regards,

Francesco