Review Reports

Round 1

Reviewer 1 Report

This review article provides a comprehensive overview of extracellular superoxide dismutase (EC-SOD, SOD3) and its role in acute respiratory distress syndrome (ARDS). The manuscript effectively synthesizes 173 references to cover EC-SOD's structural characteristics, post-translational regulation, tissue distribution, cellular sources, and genetic polymorphisms, with particular emphasis on its protective functions in acute lung injury. The integration of single-cell atlas data to illustrate cellular expression patterns represents a valuable contribution. The discussion of therapeutic strategies, including SOD mimetics and mRNA-based approaches, provides timely translational perspectives. However, several important areas require further development to provide a more complete and clinically relevant review. 

 Major comments

1. Both figures are derived from external databases rather than original experimental data. Figure 1 utilizes data from the Human Lung Cell Atlas and Neonatal Lung Atlas, while Figure 2 presents data from the COVIDome project. The authors should clarify the data extraction and visualization methods used, confirm compliance with data usage policies of these repositories, and provide appropriate acknowledgments. In Figure 1, the authors integrate data from multiple atlases across different species (human and mouse) and age groups (adult and neonatal). The comparability and limitations of this cross-species, cross-age data integration should be discussed in the figure legend or text.

2. The manuscript would be significantly strengthened by inclusion of a comprehensive schematic figure illustrating EC-SOD's regulatory network in ARDS. This figure should depict domain structure, cellular sources, pathogenic mechanisms, and therapeutic intervention points. Such an integrative figure would greatly enhance reader comprehension of this complex topic.

3. The authors report elevated plasma EC-SOD activity in ARDS patients (Ref. 116), yet decreased EC-SOD levels in SARS-CoV-2 patients (Figure 2). The current explanation attributing this discrepancy solely to disease severity and etiology is insufficient. Notably, Ref. 116 measured enzymatic activity, whereas Figure 2 utilized SOMAscan proteomics to quantify protein abundance. These fundamentally different parameters may not directly correlate, particularly under conditions of oxidative stress. A more refined analysis that explicitly accounts for methodological differences, sampling timepoints relative to disease progression, and patient heterogeneity, including differences in comorbidities and therapeutic interventions, is needed.

4. Plasma EC-SOD concentrations correlate with cardiovascular outcomes and mortality in diabetic patients (Cardiovasc Diabetol 2015;14:845). The authors should discuss whether monitoring EC-SOD levels could facilitate risk stratification or guide therapeutic interventions in ARDS patients.

5. Pulmonary hypertension represents an important complication of ARDS that is not extensively discussed in the current manuscript. EC-SOD loss of function has been shown to exacerbate pulmonary arterial hypertension (Hypertension 2011;58:303–309). Consideration of this aspect would provide a more complete picture of EC-SOD's role in ARDS-related cardiovascular complications.

6. ARDS patients frequently develop multi-organ failure, with sepsis-associated acute kidney injury occurring in up to two-thirds of septic shock patients. Given that the manuscript acknowledges sepsis as a major ARDS etiology and already extends discussion to cardiovascular disease and diabetic complications, incorporating EC-SOD's renal protective effects (J Am Soc Nephrol 2015;26:2447-2459) would provide a more comprehensive perspective on its systemic relevance in critical illness.

7. The manuscript discusses various SOD mimetics but lacks structured comparison. A summary table comparing catalytic efficiencies, tissue distributions, pharmacokinetic profiles, and safety data would significantly enhance translational utility.

8. While SOD mimetics and mRNA therapies are discussed, gene therapy is notably absent. EC-SOD gene transfer has been demonstrated to ameliorate pulmonary hypertension in animal models (Am J Respir Crit Care Med 2008;177:219-226). A brief discussion of this approach would provide a more complete overview of therapeutic strategies.

9. While Ref. 107 is cited regarding EC-SOD's effects on γδ T cells, the underlying mechanisms and clinical implications are insufficiently discussed. Given the emerging role of γδ T cells in pulmonary immunity, more detailed treatment is warranted.

10. Min et al. (Ref. 100) demonstrated synergistic protection through combined neutrophil blockade and EC-SOD overexpression. This important finding suggesting that combination therapeutic approaches may be superior to monotherapy is mentioned but not adequately discussed in Section 4.

11. The discussion of ARDS heterogeneity is absent. Current ARDS research emphasizes distinct phenotypes (hyperinflammatory versus hypoinflammatory). Discussing how EC-SOD biology might differ across these phenotypes would help identify patient subpopulations most likely to benefit from targeted therapies.

Minor issues:

1. Ref. 36 is duplicated in the citation on Line 209, appearing as "[36,36,93,94]". This error should be corrected.

2. In the Abstract, "important antioxidant enzyme" should read "an important antioxidant enzyme," and "homeostasis disease pathogenesis" should be corrected to "homeostasis and disease pathogenesis."

3. On Line 93, "heparin-birnding domain" should be corrected to "heparin-binding domain."

4. On Line 290, "because must be metabolized extracellularly b before cellular uptake" appears to contain typographical errors and should be revised for clarity.

5. On Line 384, "heparin-bidning domaine" contains spelling errors and should be corrected to "heparin-binding domain." A thorough proofreading of the entire manuscript is recommended.

6. Furin cleavage is introduced in Section 1 (Lines 84-86) as a mechanism affecting EC-SOD heparin affinity, but its regulation by genetic variants such as R213G is not discussed until Section 5 (Lines 387-388). Adding brief cross-references at the initial mention would help readers understand the clinical significance of this biochemical process.

7. In Section 3, Lines 248-249, "This process leaves the normally protected endothelium by a coating of ECSOD, vulnerable to oxidative damage" is grammatically unclear. For clarity, consider revising to: "This process leaves the endothelium, which is normally protected by a coating of EC-SOD, vulnerable to oxidative damage."

8. The authors should ensure balanced citation practices throughout the manuscript. While self-citation of relevant work is appropriate, particularly given the authors' significant contributions to the field (e.g., References 7, 41, 53, 64, 78, 113, and 114), comprehensive coverage of studies from other independent research groups investigating EC-SOD in acute lung injury models should be confirmed to ensure a truly representative synthesis of the current literature.

Author Response

1. Both figures are derived from external databases rather than original experimental data. Figure 1 utilizes data from the Human Lung Cell Atlas and Neonatal Lung Atlas, while Figure 2 presents data from the COVIDome project. The authors should clarify the data extraction and visualization methods used, confirm compliance with data usage policies of these repositories, and provide appropriate acknowledgments. In Figure 1, the authors integrate data from multiple atlases across different species (human and mouse) and age groups (adult and neonatal). The comparability and limitations of this cross-species, cross-age data integration should be discussed in the figure legend or text.

   We thank the reviewer for highlighting the need for appropriate citation, description of data extraction, and data compliance usage. The data used for generation of Figures 1 and 2 in this manuscript are open-access and made available through the online platforms: Human Lung Atlas, Neonatal single cell compressed atlas, and COVIDome Project. We did not use databases or data extraction beyond interaction with online interfaces of these databases, which are readily available to public. We provided appropriate citations and acknowledgements of all 3 resources in the Figure Legends and Data Availability Statement. We also highlighted the age-related and interspecies differences within the text of the manuscript.

 

2. The manuscript would be significantly strengthened by inclusion of a comprehensive schematic figure illustrating EC-SOD's regulatory network in ARDS. This figure should depict domain structure, cellular sources, pathogenic mechanisms, and therapeutic intervention points. Such an integrative figure would greatly enhance reader comprehension of this complex topic.

    We thank the reviewer for this suggestion. We created Figure 3 to summarize the three domains of EC-SOD and its modifications, important cellular sources of EC-SOD in the lung, the mechanisms of protection by EC-SOD against pathogenesis of acute lung injury, and therapeutic interventions.

 

3. The authors report elevated plasma EC-SOD activity in ARDS patients (Ref. 116), yet decreased EC-SOD levels in SARS-CoV-2 patients (Figure 2). The current explanation attributing this discrepancy solely to disease severity and etiology is insufficient. Notably, Ref. 116 measured enzymatic activity, whereas Figure 2 utilized SOMAscan proteomics to quantify protein abundance. These fundamentally different parameters may not directly correlate, particularly under conditions of oxidative stress. A more refined analysis that explicitly accounts for methodological differences, sampling timepoints relative to disease progression, and patient heterogeneity, including differences in comorbidities and therapeutic interventions, is needed.

    Thank you for highlighting these important differences. We have included an expanded discussion clarifying the observations of EC-SOD levels and activity.

 

4. Plasma EC-SOD concentrations correlate with cardiovascular outcomes and mortality in diabetic patients (Cardiovasc Diabetol 2015;14:845). The authors should discuss whether monitoring EC-SOD levels could facilitate risk stratification or guide therapeutic interventions in ARDS patients.

   We thank the reviewer for emphasizing this important study and for the insightful suggestion. We have now expanded the discussion to address the potential clinical implications of monitoring plasma EC-SOD levels in ARDS . Specifically, we discuss that, analogous to findings in diabetic cardiovascular disease, measurements of EC-SOD levels and reactive oxygen species may reflect endothelial and epithelial injury, processes central to ARDS pathophysiology and serve as markers of disease severity, ARDS phenotypes, and/or therapy response.

 

5. Pulmonary hypertension represents an important complication of ARDS that is not extensively discussed in the current manuscript. EC-SOD loss of function has been shown to exacerbate pulmonary arterial hypertension (Hypertension 2011;58:303–309). Consideration of this aspect would provide a more complete picture of EC-SOD's role in ARDS-related cardiovascular complications.

   We thank the reviewer for highlighting the role of EC-SOD in human pulmonary arterial hypertension and experimental chronic hypoxic pulmonary hypertension. We agree that pulmonary hypertension can occur as a short-term cardiovascular complication of ARDS and that EC-SOD–mediated redox signaling is relevant to pulmonary vascular biology. However, to the best of our knowledge, EC-SOD has not been examined in the context of pulmonary hypertension in a model of ARDS.  We felt that a detailed discussion of chronic hypoxic pulmonary hypertension was beyond the scope of the present manuscript, which is focused on the acute pathophysiology of ARDS. Based on these comments, we have noted the importance of EC-SOD and hypoxia as an important feature of ARDS

 

6. ARDS patients frequently develop multi-organ failure, with sepsis-associated acute kidney injury occurring in up to two-thirds of septic shock patients. Given that the manuscript acknowledges sepsis as a major ARDS etiology and already extends discussion to cardiovascular disease and diabetic complications, incorporating EC-SOD's renal protective effects (J Am Soc Nephrol 2015;26:2447-2459) would provide a more comprehensive perspective on its systemic relevance in critical illness.

   We thank the reviewer for highlighting the importance of sepsis as a major cause of non-pulmonary ARDS. We significantly expanded the discussion by summarizing the evidence for the protective role of EC-SOD against endothelial injury and glycocalyx degradation in sepsis and ARDS.

 

7. The manuscript discusses various SOD mimetics but lacks structured comparison. A summary table comparing catalytic efficiencies, tissue distributions, pharmacokinetic profiles, and safety data would significantly enhance translational utility.

   We created Table 1 summarizing the pharmacokinetic attributes for the SOD mimetics discussed in the manuscript.  It is important to note that the table is limited as information about half-life (t1/2) and maximum plasma concentration (Cmax) are not readily available for several of the mimetics despite these drugs being approved for clinical trials. We opted to list the phase(s) of clinical trials that have either completed or are ongoing to demonstrate the work on safety profiles of these agents.

 

8. While SOD mimetics and mRNA therapies are discussed, gene therapy is notably absent. EC-SOD gene transfer has been demonstrated to ameliorate pulmonary hypertension in animal models (Am J Respir Crit Care Med 2008;177:219-226). A brief discussion of this approach would provide a more complete overview of therapeutic strategies.

   Thank you for this important feedback regarding gene therapy as this was not adequately discussed in the initial draft. Upon closer review of the existing primary literature, EC-SOD gene therapy has been studied in an endotoxin-murine acute lung injury model, which we now discussed. We also added brief summary of about adenovirus-mediated gene transfer of sod3 in preclinical model of acute lung injury.

 

9. While Ref. 107 is cited regarding EC-SOD's effects on γδ T cells, the underlying mechanisms and clinical implications are insufficiently discussed. Given the emerging role of γδ T cells in pulmonary immunity, more detailed treatment is warranted.

   We expanded the discussion on the complex role of γδ T cells in regulating neutrophil recruitment

 

10. Min et al. (Ref. 100) demonstrated synergistic protection through combined neutrophil blockade and EC-SOD overexpression. This important finding suggesting that combination therapeutic approaches may be superior to monotherapy is mentioned but not adequately discussed in Section 4.

   Thank you for this important point. We added the discussion of the benefit of combination therapy.

 

11. The discussion of ARDS heterogeneity is absent. Current ARDS research emphasizes distinct phenotypes (hyperinflammatory versus hypoinflammatory). Discussing how EC-SOD biology might differ across these phenotypes would help identify patient subpopulations most likely to benefit from targeted therapies.

   We thank the reviewer for emphasizing the importance of phenotyping in ARDS. We agree that ARDS is a biologically heterogeneous syndrome and that emerging evidence supports the existence of distinct inflammatory phenotypes, - hyperinflammatory and hypoinflammatory. We have now expanded the discussion to address how EC-SOD biology and oxidative stress may differ across these phenotypes. We noted that direct assessments of EC-SOD across ARDS phenotypes are currently lacking, but phenotype-specific evaluation of EC-SOD and oxidative stress pathways may help identify patient subgroups that could benefit from targeted antioxidant or EC-SOD–modulating therapies

Reviewer #1 Minor Comments:

We made all appropriate edits and corrections based on the Minor Comments below. Additionally, we edited grammar and sentence structure to improve clarity and omitted sentences which distracted from the main focus of the manuscript. We also ensured a citation balance from other groups who have investigated the role of EC-SOD in acute lung injury. Less than 10 of the 170+ citations in thus manuscript originated from research our group. 

1. Ref. 36 is duplicated in the citation on Line 209, appearing as "[36,36,93,94]". This error should be corrected.
2. In the Abstract, "important antioxidant enzyme" should read "an important antioxidant enzyme," and "homeostasis disease pathogenesis" should be corrected to "homeostasis and disease pathogenesis."
3. On Line 93, "heparin-birnding domain" should be corrected to "heparin-binding domain."
4. On Line 290, "because must be metabolized extracellularly b before cellular uptake" appears to contain typographical errors and should be revised for clarity.
5. On Line 384, "heparin-bidning domaine" contains spelling errors and should be corrected to "heparin-binding domain." A thorough proofreading of the entire manuscript is recommended.
6. Furin cleavage is introduced in Section 1 (Lines 84-86) as a mechanism affecting EC-SOD heparin affinity, but its regulation by genetic variants such as R213G is not discussed until Section 5 (Lines 387-388). Adding brief cross-references at the initial mention would help readers understand the clinical significance of this biochemical process.
7. In Section 3, Lines 248-249, "This process leaves the normally protected endothelium by a coating of ECSOD, vulnerable to oxidative damage" is grammatically unclear. For clarity, consider revising to: "This process leaves the endothelium, which is normally protected by a coating of EC-SOD, vulnerable to oxidative damage."
8. The authors should ensure balanced citation practices throughout the manuscript. While self-citation of relevant work is appropriate, particularly given the authors' significant contributions to the field (e.g., References 7, 41, 53, 64, 78, 113, and 114), comprehensive coverage of studies from other independent research groups investigating EC-SOD in acute lung injury models should be confirmed to ensure a truly representative synthesis of the current literature.

Reviewer 2 Report

The authors present strong preclinical data but do not explain why EC-SOD has never been tested in human ARDS trials - that gap needs discussion. The conflicting plasma EC-SOD data between studies is confusing and needs better explanation than just guessing about disease severity. Most animal studies use pretreatment before injury, but real patients arrive already sick, so the clinical relevance is unclear.

1. The manuscript lacks clarity on the clinical translation gap. While the preclinical data showing EC-SOD's protective effects are comprehensive, the authors do not adequately address why these findings have not beenn translated to clinical trials in humans with ARDS. The authors mention "no data regarding rhEC-SOD in human patients" but do not discuss the barriers preventing such trials or propose specific strategies to overcome them.

2. Please clarify the conflicting data on plasma EC-SOD levels in ARDS patients (elevated in one study, reduced in COVID-19 patients). The authors suggest disease severity and etiology may explain differences, but this remains speculative. A more thorough discussion of how different ARDS etiologies might affect EC-SOD expression and distribution would strengthen the manuscript.

3. The section on therapeutic strategies would benefit from discussing optimal timing and delivery methods more explicitly. Most preclinical studies used pretreatment protocols, which is not clinically realistic for ARDS. The manuscript should address whether post-injury administration of EC-SOD or SOD mimetics has shown efficacy and what this means for clinical application.

4. The discussion of genetic polymorphisms, while thorough, does not connect well to the ARDS focus. The R213G variant section notes the study was underpowered for ARDS outcomes, yet considerable space is devoted to associations with other conditions. Either this section should be condensed or the authors should propose how understanding these variants could guide personalized ARDS therapy.

5. The manuscript would benefit from a dedicated section on knowledge gaps and future research priorities. Currently, these are scattered throughout. Specifically, the potential intracellular functions of EC-SOD, the mechanisms regulating its redistribution during inflammation, and the discrepancy between animal model success and clinical trial failures all warrant systematic discussion.

Author Response

1. The manuscript lacks clarity on the clinical translation gap. While the preclinical data showing EC-SOD's protective effects are comprehensive, the authors do not adequately address why these findings have not beenn translated to clinical trials in humans with ARDS. The authors mention "no data regarding rhEC-SOD in human patients" but do not discuss the barriers preventing such trials or propose specific strategies to overcome them.

   We thank the reviewer for raising this important point regarding the translational gap between preclinical EC-SOD and antioxidant studies and failure to show benefit in the human ARDS trials. We agree that, despite robust protective effects observed in experimental models, several barriers have limited clinical translation to date. These include challenges related to drug delivery to the injured lung, uncertainty regarding optimal timing and dosing in the rapidly evolving course of ARDS, concerns about off-target effects of systemic antioxidant therapy, and the biological heterogeneity of ARDS, which complicates patient selection for interventional trials. In addition, recombinant human EC-SOD has limited clinical availability and has not been extensively developed for acute critical illness indications. We have revised the discussion to explicitly address these barriers and to clarify the current limitations.

 

2. Please clarify the conflicting data on plasma EC-SOD levels in ARDS patients (elevated in one study, reduced in COVID-19 patients). The authors suggest disease severity and etiology may explain differences, but this remains speculative. A more thorough discussion of how different ARDS etiologies might affect EC-SOD expression and distribution would strengthen the manuscript.

    Thank you for highlighting these important differences. We have included an expanded discussion clarifying the observations of EC-SOD levels, activity, and limitations in the revised manuscript.

 

3. The section on therapeutic strategies would benefit from discussing optimal timing and delivery methods more explicitly. Most preclinical studies used pretreatment protocols, which is not clinically realistic for ARDS. The manuscript should address whether post-injury administration of EC-SOD or SOD mimetics has shown efficacy and what this means for clinical application.

   Thank you for this important point. We have expanded the discussion to address timing of administration and explored the prophylactic use of SOD mimetics in patients at risk for developing ARDS.

 

4. The discussion of genetic polymorphisms, while thorough, does not connect well to the ARDS focus. The R213G variant section notes the study was underpowered for ARDS outcomes, yet considerable space is devoted to associations with other conditions. Either this section should be condensed or the authors should propose how understanding these variants could guide personalized ARDS therapy.

  We thank the reviewer for this constructive comment. We have edited this section to emphasize the translational potential of EC-SOD genetics while acknowledging limitations of current evidence.

 

5. The manuscript would benefit from a dedicated section on knowledge gaps and future research priorities. Currently, these are scattered throughout. Specifically, the potential intracellular functions of EC-SOD, the mechanisms regulating its redistribution during inflammation, and the discrepancy between animal model success and clinical trial failures all warrant systematic discussion.

   We thank the reviewer for this thoughtful suggestion and agree that identifying knowledge gaps and future research priorities is important. In the current manuscript, we intentionally integrated discussion of unresolved questions and translational challenges within the relevant sections, rather than presenting them in a standalone section, to maintain narrative flow and avoid redundancy. The intracellular actions of EC-SOD, mechanisms governing its redistribution during inflammation, and the discrepancy between preclinical success and limited clinical translation are each addressed at the points where the corresponding biology or experimental evidence is discussed. We believe this integrated approach allows these knowledge gaps to be considered in direct context with the available data, while preserving the focus and organization of this review.

Round 2

Reviewer 1 Report

The authors have adequately addressed the concerns raised in the previous review. They have significantly strengthened the manuscript by including a new comprehensive schematic illustrating the regulatory network, domain structure, and therapeutic intervention points of EC-SOD in ARDS (Figure 3, Pages 13-14). A new summary table comparing the pharmacokinetic profiles and clinical trial status of various SOD mimetics has also been added (Table 1, Page 14), enhancing the translational utility of the review. The discussion has been expanded to cover ARDS phenotypes and their differential relevance to EC-SOD biology (Page 8, Lines 344-354). Furthermore, the manuscript now provides a clearer explanation for the apparent discrepancy between plasma EC-SOD activity and protein levels by accounting for proteolytic cleavage and cell lysis mechanisms (Page 8, Lines 328-343). The data sources for Figures 1 and 2 have been appropriately clarified in the figure legends (Page 4, Lines 164-166; Page 9, Lines 361-362) and Data Availability Statement (Page 14, Lines 570-572). Additionally, all Minor Issues have been appropriately corrected, including spelling errors, grammatical issues, duplicate citations, and the addition of cross-references linking Arg213 to the R213G variant discussion.

However, the manuscript in its current form due to severe and pervasive citation errors in the newly added sections. A detailed verification of the revised text reveals that multiple reference numbers do not correspond to the cited literature in the bibliography, rendering these citations scientifically inaccurate. The authors must rigorously verify all citations in the manuscript to ensure they match the text content, and this verification process should extend to all revisions made, including citations added in response to other reviewers, not just the errors identified here. The manuscript should not proceed to publication until these significant bibliographic inaccuracies are comprehensively corrected.

The authors have adequately addressed the concerns raised in the previous review. They have significantly strengthened the manuscript by including a new comprehensive schematic illustrating the regulatory network, domain structure, and therapeutic intervention points of EC-SOD in ARDS (Figure 3, Pages 13-14). A new summary table comparing the pharmacokinetic profiles and clinical trial status of various SOD mimetics has also been added (Table 1, Page 14), enhancing the translational utility of the review. The discussion has been expanded to cover ARDS phenotypes and their differential relevance to EC-SOD biology (Page 8, Lines 344-354). Furthermore, the manuscript now provides a clearer explanation for the apparent discrepancy between plasma EC-SOD activity and protein levels by accounting for proteolytic cleavage and cell lysis mechanisms (Page 8, Lines 328-343). The data sources for Figures 1 and 2 have been appropriately clarified in the figure legends (Page 4, Lines 164-166; Page 9, Lines 361-362) and Data Availability Statement (Page 14, Lines 570-572). Additionally, all Minor Issues have been appropriately corrected, including spelling errors, grammatical issues, duplicate citations, and the addition of cross-references linking Arg213 to the R213G variant discussion.

However, the manuscript in its current form due to severe and pervasive citation errors in the newly added sections. A detailed verification of the revised text reveals that multiple reference numbers do not correspond to the cited literature in the bibliography, rendering these citations scientifically inaccurate. For example, the text states that adenovirus-mediated gene transfer of sod3 protects against lung injury (Page 11, Lines 459-461) and cites Ref. [128], but Ref. [128] is titled "Oxidized Glutathione Is Increased in the Alveolar Fluid..." which describes glutathione metabolism and has no relation to adenovirus gene transfer. Similarly, the discussion on the synergistic potential of Antileukinate and EC-SOD (Page 11, Lines 467-472) cites Ref. [129], which is titled "Mechanism of Action of Vitamin C in Sepsis" and is factually incorrect for the context. The section on the immunological role of gamma-delta T cells (Page 7, Lines 289-292) cites Refs. [141-143], but a review of the bibliography shows that these references discuss SOD mimetics (Desferrioxamine, Salen-Mn) rather than T-cell immunology. Furthermore, regarding the renal protective effects discussed in response to Major Comment #6, the authors cite Refs. (p. 6, lines 224–228) [113–116]. In this context, these studies primarily address lung injury, thrombosis, or general oxidative stress and do not investigate glycocalyx or heparan sulfate–related mechanisms, rendering them irrelevant to the discussion of glycocalyx degradation in this section. Moreover, the revised discussion (p. 6, lines 224–228) shifts the focus away from the antioxidant role of EC-SOD itself. Notably, direct evidence for EC-SOD–mediated organ protection under septic conditions has been reported (e.g., J Am Soc Nephrol 2015;26:2447–2459), yet this mechanistic framework is not adequately addressed. As a result, the cited literature does not sufficiently support the stated claims, and a more appropriate, topic-aligned discussion is required.

In addition, a targeted review of other sections in the V2 manuscript identifies further citation inconsistencies that have not been exhaustively itemized above. Specifically, in the Therapeutics section, statements regarding N-acetylcysteine and Vitamin C clinical trials (Page 10, Lines 368-369) cite Refs. [165–167]; however, these reference numbers correspond in the bibliography to studies on EC-SOD genetic polymorphisms and diabetic complications rather than interventional or clinical trial data. Likewise, multiple citations in the later Polymorphisms section refer to reference numbers exceeding the terminal entry of the bibliography (e.g., Refs. [189–192] cited for the R213G variant on Page 11, Lines 479–481), indicating the presence of non-existent or “ghost” citations. These additional findings further support the conclusion that citation misalignment in the revised manuscript extends beyond the specific examples discussed above and affects multiple sections introduced or modified in V2.

The authors must rigorously verify all citations in the manuscript to ensure they match the text content, and this verification process should extend to all revisions made, including citations added in response to other reviewers, not just the errors identified here. The manuscript should not proceed to publication until these significant bibliographic inaccuracies are comprehensively corrected.

Author Response

Reviewer #1 V2/Revision Comments

The authors have adequately addressed the concerns raised in the previous review. They have significantly strengthened the manuscript by including a new comprehensive schematic illustrating the regulatory network, domain structure, and therapeutic intervention points of EC-SOD in ARDS (Figure 3, Pages 13-14). A new summary table comparing the pharmacokinetic profiles and clinical trial status of various SOD mimetics has also been added (Table 1, Page 14), enhancing the translational utility of the review. The discussion has been expanded to cover ARDS phenotypes and their differential relevance to EC-SOD biology (Page 8, Lines 344-354). Furthermore, the manuscript now provides a clearer explanation for the apparent discrepancy between plasma EC-SOD activity and protein levels by accounting for proteolytic cleavage and cell lysis mechanisms (Page 8, Lines 328-343). The data sources for Figures 1 and 2 have been appropriately clarified in the figure legends (Page 4, Lines 164-166; Page 9, Lines 361-362) and Data Availability Statement (Page 14, Lines 570-572). Additionally, all Minor Issues have been appropriately corrected, including spelling errors, grammatical issues, duplicate citations, and the addition of cross-references linking Arg213 to the R213G variant discussion.

However, the manuscript in its current form due to severe and pervasive citation errors in the newly added sections. A detailed verification of the revised text reveals that multiple reference numbers do not correspond to the cited literature in the bibliography, rendering these citations scientifically inaccurate. For example, the text states that adenovirus-mediated gene transfer of sod3 protects against lung injury (Page 11, Lines 459-461) and cites Ref. [128], but Ref. [128] is titled "Oxidized Glutathione Is Increased in the Alveolar Fluid..." which describes glutathione metabolism and has no relation to adenovirus gene transfer. Similarly, the discussion on the synergistic potential of Antileukinate and EC-SOD (Page 11, Lines 467-472) cites Ref. [129], which is titled "Mechanism of Action of Vitamin C in Sepsis" and is factually incorrect for the context. The section on the immunological role of gamma-delta T cells (Page 7, Lines 289-292) cites Refs. [141-143], but a review of the bibliography shows that these references discuss SOD mimetics (Desferrioxamine, Salen-Mn) rather than T-cell immunology. Furthermore, regarding the renal protective effects discussed in response to Major Comment #6, the authors cite Refs. (p. 6, lines 224–228) [113–116]. In this context, these studies primarily address lung injury, thrombosis, or general oxidative stress and do not investigate glycocalyx or heparan sulfate–related mechanisms, rendering them irrelevant to the discussion of glycocalyx degradation in this section. Moreover, the revised discussion (p. 6, lines 224–228) shifts the focus away from the antioxidant role of EC-SOD itself. Notably, direct evidence for EC-SOD–mediated organ protection under septic conditions has been reported (e.g., J Am Soc Nephrol 2015;26:2447–2459), yet this mechanistic framework is not adequately addressed. As a result, the cited literature does not sufficiently support the stated claims, and a more appropriate, topic-aligned discussion is required.

In addition, a targeted review of other sections in the V2 manuscript identifies further citation inconsistencies that have not been exhaustively itemized above. Specifically, in the Therapeutics section, statements regarding N-acetylcysteine and Vitamin C clinical trials (Page 10, Lines 368-369) cite Refs. [165–167]; however, these reference numbers correspond in the bibliography to studies on EC-SOD genetic polymorphisms and diabetic complications rather than interventional or clinical trial data. Likewise, multiple citations in the later Polymorphisms section refer to reference numbers exceeding the terminal entry of the bibliography (e.g., Refs. [189–192] cited for the R213G variant on Page 11, Lines 479–481), indicating the presence of non-existent or “ghost” citations. These additional findings further support the conclusion that citation misalignment in the revised manuscript extends beyond the specific examples discussed above and affects multiple sections introduced or modified in V2.

The authors must rigorously verify all citations in the manuscript to ensure they match the text content, and this verification process should extend to all revisions made, including citations added in response to other reviewers, not just the errors identified here. The manuscript should not proceed to publication until these significant bibliographic inaccuracies are comprehensively corrected.

We thank the reviewer for vigorous review of the manuscript and cited sources. We reviewed and corrected each individual citations and ensured that citations correspond to appropriate text within the manuscript.

In this context, these studies primarily address lung injury, thrombosis, or general oxidative stress and do not investigate glycocalyx or heparan sulfate–related mechanisms, rendering them irrelevant to the discussion of glycocalyx degradation in this section. Moreover, the revised discussion (p. 6, lines 224–228) shifts the focus away from the antioxidant role of EC-SOD itself. Notably, direct evidence for EC-SOD–mediated organ protection under septic conditions has been reported (e.g., J Am Soc Nephrol 2015;26:2447–2459), yet this mechanistic framework is not adequately addressed. As a result, the cited literature does not sufficiently support the stated claims, and a more appropriate, topic-aligned discussion is required.

We thank the reviewer for this insightful critique. We agree that the previous discussion lacked the specific mechanistic relationship between EC-SOD and the endothelial glycocalyx. In the revised manuscript, we have refocused the discussion on the heparin-binding domain of EC-SOD, which allows it to localize specifically within the glycocalyx layer to protect heparan sulfate fragments from oxidative cleavage. We also incorporated the suggested literature, specifically citing the work in J Am Soc Nephrol (2015) to demonstrate how EC-SOD-mediated protection is vital in septic conditions, particularly in renal injury.

Reviewer 2 Report

The authors addressed all my comments in their revised manuscript.

The authors addressed all my comments in their revised manuscript.

Author Response

Reviewer #2 V2/Revision Comments:

The authors addressed all my comments in their revised manuscript.

Round 3

Reviewer 1 Report

The authors have successfully corrected the pervasive citation misalignments identified in the previous version. Specifically, they have re-aligned references for adenovirus-mediated gene transfer (corrected from 128 to 149), Antileukinate synergy (corrected from 129 to 150), and gamma-delta T-cell immunology (corrected from 141–143 to 162–164). Crucially, the requested mechanistic framework for renal protection has been integrated by incorporating key evidence (Ref. 135), focusing on the interaction between the EC-SOD heparin-binding domain and the endothelial glycocalyx. Furthermore, the addition of corresponding references within the tables (Table 1) significantly enhances the manuscript's utility, providing readers with a convenient and reliable resource for cross-referencing. The previously identified 'ghost citations' have been resolved by re-indexing and expanding the bibliography to 239 entries, making the manuscript scientifically sound and recommended for publication.

The authors have successfully corrected the pervasive citation misalignments identified in the previous version. Specifically, they have re-aligned references for adenovirus-mediated gene transfer (corrected from 128 to 149), Antileukinate synergy (corrected from 129 to 150), and gamma-delta T-cell immunology (corrected from 141–143 to 162–164). Crucially, the requested mechanistic framework for renal protection has been integrated by incorporating key evidence (Ref. 135), focusing on the interaction between the EC-SOD heparin-binding domain and the endothelial glycocalyx. Furthermore, the addition of corresponding references within the tables (Table 1) significantly enhances the manuscript's utility, providing readers with a convenient and reliable resource for cross-referencing. The previously identified 'ghost citations' have been resolved by re-indexing and expanding the bibliography to 239 entries, making the manuscript scientifically sound and recommended for publication.