Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses and revisions below. All line numbers refer to the line-numbered revised manuscript submitted alongside this response.

2. Questions for General Evaluation

Question

Reviewer's Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Addressed. New multi-framework paragraph (lines 72–87) cites 10 independent research groups. Additional, 12 new independent references added across this R2 revision.

Are all the cited references relevant to the research?

Can be improved

Addressed. Three self-citations removed (former refs 47, 49, 50). Self-citation rate: 15/71 = 21.1%.

Is the research design appropriate?

Yes

No action required.

Are the methods adequately described?

Yes

No action required.

Are the results clearly presented?

Can be improved

Addressed. Table 2 completely redesigned as neutral three-column modality comparison. Subheadings standardised throughout.

Are the conclusions supported by the results?

Yes

No action required.

3. Point-by-Point Response to Comments and Suggestions for Authors

Comment 1:

"Asif Ahmed's group were pioneers in development of the two-hit hypothesis over the past thirty years, but other groups, who are not cited in this review, also were involved... The number of references in which Asif Ahmed is first author would in itself be considered excessive self-citation."

Response 1:

Addressed. We accept this criticism. The following substantive changes have been made:

(i) New contextualising paragraph (lines 72-87). Epidemiology and Clinical Burden section. Ten independent research groups are now cited: defective trophoblast invasion (Pijnenborg et al. [3]; Fisher [4]); complement dysregulation (Lynch & Salmon [5]; Burwick 2025 [6]); immune maladaptation (Redman & Sargent [7, 8]); renin-angiotensin autoantibodies (Wallukat [9]; Hubel [10]); and mechanistic vascular effects (Dechend [11]; Irani & Xia [12]).

(ii) Placental VEGF Signalling section (line 143): language depersonalised. "In 2001, Ahmed's group showed" replaced with "Early studies demonstrated." The VEGF loss hypothesis is now described in the passive voice in the lines 113-118.

(iii) Table 1: parenthetical "(Ahmed)" removed from the 1997 entry. Attribution is provided through citation [13] in the References column.

(iv) HO-1 section (line 201): first-person corrected to third person. Text now reads: "Ahmed et al. (2000) demonstrated, for the first time in human placental tissue, that HO-1 induction protects against cytokine-mediated cytotoxicity."

(v) Three self-citations removed. Former refs 47, 49, 50 (see Editorial Office response). Revised self-citation rate: 15/71 = 21.1%.

Note on Conceptual Evolution section (lines 459-477): this section explicitly traces the intellectual history of the double-hit hypothesis originating from this laboratory. The self-citations retained here ([59], [60]) are the primary historical records of that development and cannot be replaced by independent references.

Comment 2:

"How does hypoxia cause excess placental sFlt-1 production, which antagonizes VEGF activity, when hypoxia normally results in increased VEGF production?"

Response 2:

Addressed. New section "Hypoxia, HIF Signalling, and sFlt-1 Regulation" added at lines 120-146.

The section explains: hypoxia stabilises HIF-1α and HIF-2α in trophoblasts, enabling transcriptional upregulation of both VEGFA and FLT1 [20]. HIF-2α (not HIF-1α) is the dominant driver of sFlt-1 upregulation in trophoblasts, silencing HIF-2α attenuates sFlt-1 expression (Sasagawa et al. 2018 [21]). Although local VEGF expression increases, preferential secretion of the soluble receptor isoform shifts the systemic balance toward VEGF neutralisation (Nevo et al. 2006 [22]; Levine et al. [23]; Maynard et al. [24]), reducing circulating free VEGF bioactivity. This resolves the apparent paradox. Cell-type-specific post-transcriptional regulation by hnRNP D provides a further regulatory layer [25].

Comment 3:

"How HO-1, CSE, and carbon monoxide and H₂S signaling act as a protective brake and how it fails are inadequately described."

Response 3:

Addressed. The Cytoprotective Pathways section (Hit 2) has been substantially expanded at lines 194-350.

The expanded mechanistic paragraph now covers:

• HO-1/CO axis (lines 330–337): HO-1 induction suppresses sFlt-1 and sEng release through CO-dependent signalling and modulation of redox-sensitive transcriptional pathways [29]. CO enhances endothelial NO bioavailability and limits inflammatory activation, stabilising endothelial function under angiogenic stress. Bilirubin (via HO-1/BVR) acts as a lipid-phase antioxidant, reducing oxidative amplification of anti-angiogenic signalling.

• CSE/H₂S axis (lines 339–344): H₂S promotes vasodilation, preserves mitochondrial bioenergetics, and suppresses ROS accumulation. CSE inhibition experimentally increases placental sFlt-1 and sEng; H₂S donors restore angiogenic balance in pregnancy models [30]. At the endothelial level, H₂S supports NO signalling and enhances resistance to VEGF withdrawal-induced dysfunction [49].

• Double-hit integration (lines 346-350): "Failure of these cytoprotective systems lowers the threshold at which placental anti-angiogenic stress translates into systemic endothelial decompensation. Hit 2 amplifies the biological consequences of Hit 1 rather than initiating placental factor release itself."

Comment 4:

"The MZe786 compound is the focus of Table 2, which seems more appropriate for a grant application or investment prospectus than a scholarly review."

Response 4:

Addressed. Table 2 has been completely redesigned.

The original two-column, MZe786-focused format has been replaced with a neutral three-column comparison of therapeutic modality classes: (1) oral small-molecule pathway modulator; (2) siRNA/oligonucleotide targeting sFlt-1; (3) recombinant biologic protein therapy. MZe786 is not named anywhere within the table body. The caption explicitly states that no comparative superiority is implied and that clinical trial data remain limited. Two new rows on iron/redox interface and ferroptotic susceptibility have been added in response to Reviewer 3.

Comment 5:

"The dark blue header row in the tables makes it difficult to read the titles. The subheadings in Section 3.5 are not consistent or properly presented."

Response 5:

Addressed.

Table header shading in both Table 1 and Table 2 has been revised to a mid-blue scheme with white text that is clearly readable. All second-level subheadings throughout the manuscript have been reviewed and standardised for consistent capitalisation and formatting. Final typographic consistency will be confirmed at proof stage.

4. Response to Comments on the Quality of English Language

No specific comments on English were raised. General language editing applied throughout; all revisions verified by authors.

5. Additional Clarifications

Reviewer 1 raised general concern about self-citation clustering in the Conceptual Evolution section. Refs [59] and [60] are retained there because that section explicitly traces the intellectual development of the double-hit hypothesis originating from this laboratory; they are primary historical records and cannot be replaced. All other feasible self-citation reductions have been made.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses and revisions below. All line numbers refer to the line-numbered revised manuscript submitted alongside this response.

2. Questions for General Evaluation

Question

Reviewer's Evaluation

Response and Revisions

Is the work a significant contribution to the field?

Yes

Is the work well organised and comprehensively described?

Can be improved

Addressed. Three new substantive sections added; 10 + 12 new independent references integrated.

Is the work scientifically sound and not misleading?

Yes

Are there appropriate and adequate references to related and previous work?

Can be improved

Addressed. 10 + 12 new references added; 9 published since 2020. Self-citation rate reduced to 21.1%.

Is the English used correct and readable?

Yes

3. Point-by-Point Response to Comments and Suggestions for Authors

Comment 1:

"This is an okay written review where authors have put together literature on Preeclampsia, by suggesting it as a double-hit vascular disorder with the focus on the VEGF-HO-1-CSE axis. However, the review including abstract and the paper needs significant revision. I suggest that much more recent papers - within the last 5 years - are essential to substantially improve the quality of the review."

Response 1:

Addressed. The reference base has been substantially expanded with a focus on 2020–2025 publications.

New additions across this revision (12 independent references, 9 from 2020–2025):

• Epidemiology section, line ~74: Burwick et al. 2025 [6] complement dysregulation in preeclampsia.

• HO-1 section (lines 212-226): Park et al. Circ Res 2025 [43] placental hypoxia-induced ferroptosis; Zhang et al. Int J Mol Sci 2022 [44] iron metabolism and ferroptosis in pathological pregnancy.

• Ancestral variation (lines 367-376): Wright et al. BJOG 2023 [63]; Saad et al. J Appl Lab Med 2025 [64] sFlt-1/PlGF reference ranges in diverse cohorts.

• NEW SECTION. Nitric Oxide Bioavailability (lines 239-265): Bussolati et al. 2001 [47]; Choi & Kim Front Cardiovasc Med 2021 [48]; Szijártó et al. Hypertension 2018 [49]; Guerby et al. Redox Biol 2021 [50]; Kaihara et al. Diseases 2023 [51].

• NEW SECTION. Aspirin Prophylaxis (lines 267-295): Huang et al. Hypertension 2025 [52]; Rolnik et al. NEJM 2017 [53]; Henderson et al. JAMA 2021 [54]; Henderson et al. Ann Intern Med 2014 [55]; Roberge et al. AJOG 2018 [56]; Ghesquiere et al. AJOG MFM 2023 [57]; Ngwira et al. Hypertension 2025 CASPER [58].

• DM199 section (lines 461-433): Cluver et al. BMJ Open 2025 [68]; ClinicalTrials.gov NCT06875141 [69]; Schmaier 2003 [66]; Kakoki & Smithies 2009 [67].

Total references in revised manuscript: 71. Original had just 38 references. Total new independent references added in this revision R2: 12. Total published 2020 or later: 9.

4. Response to Comments on the Quality of English Language

No specific comments on English were raised. General language editing applied throughout.

5. Additional Clarifications

This revision adds additional 12 new independent references, 9 published since 2020. The total reference list is now 71. We believe the expanded reference base comprehensively addresses the reviewer's concern about recency and breadth of coverage.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses and revisions below. All line numbers refer to the line-numbered revised manuscript submitted alongside this response.

2. Questions for General Evaluation

Question

Reviewer's Evaluation

Response and Revisions

Is the work a significant contribution to the field?

Yes

Is the work well organised and comprehensively described?

Can be improved

Addressed. Iron metabolism and ferroptosis integrated at lines 214-228 with two new rows added to Table 2.

Is the work scientifically sound and not misleading?

Yes

Are there appropriate and adequate references to related and previous work?

Can be improved

Addressed. Both recommended references (Park et al. 2025 [43]; Zhang et al. 2022 [44]) incorporated with full mechanistic context. (see lines 221-225).

Is the English used correct and readable?

Yes

3. Point-by-Point Response to Comments and Suggestions for Authors

Comment 1:

"Without any significant attention being paid to the important roles of iron and iron related processes, in particular the roles of hypoxia, hepcidin, HO-1, ferroportin, ferritin in relation to ferroptosis in the endothelium — all regulated by interactive processes with those described in this review — there is a significant gap in the review."

Response 1:

Addressed. A new paragraph integrating iron metabolism and ferroptosis within the double-hit framework has been added in the section "Heme Oxygenase-1: A Master Cytoprotective Brake", at lines 214-228.

The new paragraph covers:

(i) Iron handling at the HO-1 nexus (lines 214-228): Heme degradation liberates ferrous iron, inducing ferritin expression and modulating ferroportin-mediated iron export. Hepcidin regulates systemic iron trafficking; dysregulation of this axis may increase labile iron pools and lipid peroxidation.

(ii) Park et al. Circ Res 2025 [43] (lines 214-228): Placental hypoxia induces ferroptosis characterised by iron accumulation, glutathione depletion, reduced GPx4 activity, and lipid peroxidation, with extrusion of iron-rich, lipid peroxide-laden small extracellular vesicles into the maternal circulation, contributing to endothelial activation and impaired angiogenesis.

(iii) Zhang et al. Int J Mol Sci 2022 [44] (lines 214-228): Broader evidence implicating ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, in pathological pregnancy states.

(iv) Double-hit integration (lines 263-267): "The failure of HO-1-mediated cytoprotective buffering may amplify both anti-angiogenic signalling and iron-dependent oxidative injury, integrating redox and angiogenic pathways within the double-hit framework."

Two new rows (Iron/redox interface and Effect on ferroptotic susceptibility) have been added to Table 2 to reflect this biology across therapeutic modality classes.

Comment 2:

"See Park C et al., Circ Res 2025 and Zhang Y et al., Int J Mol Sci 2022."

Response 2:

Addressed. Both references incorporated:

Park C et al. Placental Hypoxia-Induced Ferroptosis Drives Vascular Damage in Preeclampsia. Circ Res. 2025;136(4):361–378. doi: 10.1161/CIRCRESAHA.124.325119. Now cited as [43] at line ~222.

Zhang Y, Lu Y, Jin L. Iron Metabolism and Ferroptosis in Physiological and Pathological Pregnancy. Int J Mol Sci. 2022;23(16). doi: 10.3390/ijms23169395. Now cited as [44] at line ~224.

4. Response to Comments on the Quality of English Language

No specific comments on English were raised.

5. Additional Clarifications

We thank Reviewer 3 for identifying the iron/ferroptosis gap. The integration of this biology substantially strengthens the double-hit framework by providing a mechanistically coupled downstream amplification loop at the HO-1 nexus linking both hits to iron-dependent oxidative injury.