Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2 · 10.1371/journal.pone.0046720

Name: Verified Rigor Record — Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2 · 10.1371/journal.pone.0046720
Item: Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2 · 10.1371/journal.pone.0046720
Rating: 75
Author: Αlpha¹

Paper source

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Interventional Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: highReviewer agreement: moderateEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.
No data or code availability links were detected to verify.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper's scientific premise and ethical approvals are strong, and the methods are reasonably detailed. However, multiple rigor gaps—missing randomization, blinding, power analysis, data availability, and statistical-reporting details—warrant caution. Two retracted and one unfindable reference further erode confidence.

All eight dimensions were scored. The statistics verification component found no test that could be recomputed (all p-values are threshold-only, so coverage note applies). Two retracted references and one reference not found in any registry were identified by the citation checker.

12 major claims checked against the paper's own evidence: all adequately supported.

2 integrity concerns flagged (0 high).

Rigor

Eight Dimensions3 pass · 4 warn · 1 fail

12 major claims1 partial11 supported

The introduction thoroughly cites prior work establishing that VEGFR2 is reduced in diabetes and that methylglyoxal (MGO) is implicated in diabetic complications. It then explicitly states the gap: 'The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known.' (last paragraph of introduction). The study's objectives are logically derived from this gap. Limitations of prior work are indirectly addressed by noting that many mechanisms have been proposed but the picture remains incomplete, and the study aims to address this.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedINADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The study's scientific premise is well-established through a comprehensive review of relevant literature and a clear logical rationale.

Evidence

direct quote[Introduction, paragraph 2 and 3]

“VEGFR2 protein reduction has been observed in patients with diabetes, and in experimental diabetic animals.”

direct quote[Introduction, last paragraph]

“The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known.”

direct quote[Introduction, paragraph 1]

“Multiple molecular mechanisms have been proposed, including oxidative stress/reactive oxygen species, endothelial derangements, loss of endothelium-derived nitric oxide (NO) bioactivity, and micro RNA alterations.”

PASSReviewer 2· 95% conf

The paper establishes a clear scientific premise by citing prior work on diabetic angiogenesis impairment, VEGFR2 function, and methylglyoxal/AGEs, and provides a logical rationale linking these to the study hypothesis.

Evidence

paraphrase[Introduction, paragraphs 1-4]

“The introduction cites multiple references on diabetic angiogenesis impairment, VEGFR2 signaling, and the involvement of MGO and AGEs in diabetic complications.”

direct quote[Introduction, last sentence]

“Provided the crucial role of VEGFR2 in endothelial angiogenesis and the implications of MGO in diabetic complications, this study investigated the impacts of MGO on VEGFR2 protein levels and endothelial angiogenesis in cell culture and mouse models to define a potentially new mechanism underlying angiogenesis impairment in diabetes.”

direct quote[Introduction, paragraph 3]

“The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known.”

PASSReviewer 3· 95% conf

The paper establishes a clear scientific premise by citing prior work on diabetic angiogenesis impairment, VEGFR2 reduction, and methylglyoxal (MGO) as a precursor of AGEs, and provides a logical rationale linking these to the study's objectives.

Evidence

direct quote[Introduction, paragraphs 1-4]

“Mechanisms underlying diabetic angiogenesis impairment are complex... However, it has not been completely elucidated how diabetes impairs physiological angiogenesis.”

direct quote[Introduction, last paragraph]

direct quote[Discussion, paragraph 1]

“The present study provided evidence to demonstrate that MGO... impairs endothelial angiogenesis... through ONOO– dependent and autophagy-mediated VEGFR2 degradation... suggesting a previously unidentified mechanism for angiogenesis impairment in diabetes.”

The study uses cell culture, ex vivo aortic ring assays, and in vivo mouse models with pharmacological and genetic interventions. Randomization methods and units are not reported for any experiments. Blinding is not mentioned. No a priori power analysis is provided. The distinction between biological and technical replicates is not explicitly defined, though 'n=3' is used for cell experiments and 'n=5' for aortic ring assays, suggesting biological replicates. Inclusion/exclusion criteria for mice are not pre-specified. Outlier handling is not described. Controls are adequate (e.g., vehicle controls, control siRNA, empty plasmids). Independent replication is not reported or planned. The study is a single investigation without a separate validation cohort.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionINADEQUATE

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The study design is rigorous in many aspects but has key omissions: randomization, blinding, and power analysis are not reported for the in vivo or ex vivo experiments.

Evidence

absence[Mice section]

Randomization method is not reported.

absence[Throughout Methods]

Blinding is not reported.

direct quote[Figure legends (e.g., Figure 1)]

“All blots shown are representative of three independent experiments.”

WARNReviewer 2· 80% conf

The study design lacks critical rigor elements: no randomization method, blinding, power analysis, or pre-specified inclusion/exclusion criteria are reported. Replicate distinction is implicit but not explicitly defined. Controls are adequate.

Evidence

absence[Methods, Mice section]

No randomization method or unit is described for animal treatment.

absence[Methods, statistical analysis]

No blinding of investigators or assessors is reported.

direct quote[Results and Figure legends (e.g., Figure 1 legend)]

“All blots shown are representative of three independent experiments.”

WARNReviewer 3· 85% conf

The study design is largely appropriate for a preclinical interventional study, but several key elements are not reported or are inadequately described, including randomization, blinding, power analysis, and pre-specified inclusion/exclusion criteria.

Evidence

absence[Materials and Methods]

Randomization method and unit are not reported.

absence[Materials and Methods]

Blinding is not mentioned.

direct quote[Materials and Methods, Results]

“Transfection of control or target (LC3B, Beclin-1, and Glo1) siRNA was performed based on protocols provided by Santa Cruz Biotechnology... Adenoviral infection of GFP, SOD1, and SOD2 were performed as previously reported.”

For mice, the paper states: 'Male C57BL/6J mice as well as genetic diabetic Akita and db/db (and control lean) mice, 8 weeks of age, 20–30 g, were obtained from the Jackson Laboratory.' Sex is reported as male, but no scientific justification for using only males is given. Species, strain, and source are adequately reported. For cell lines (BAEC, HUVEC), the source (ATCC) is stated, but age/weight/health are not applicable. Housing conditions (light cycle, temperature, enrichment) are not described for the mice. Demographics are not applicable (non-human).

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedNOT REPORTED

age weight healthADEQUATE

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Biological variables are partially reported: age, weight, sex, and species/strain are given for mice, but housing conditions are missing, and for cell lines, age and health status are not applicable.

Evidence

direct quote[Mice section]

“Male C57BL/6J mice as well as genetic diabetic Akita and db/db (and control lean) mice”

absence[Mice section]

Housing conditions are not reported.

direct quote[Mice section]

“Male C57BL/6J mice as well as genetic diabetic Akita and db/db (and control lean) mice, 8 weeks of age, 20–30 g, were obtained from the Jackson Laboratory”

PASSReviewer 2· 85% conf

Key biological variables for the animal studies are reported, including species, strain, sex, age, weight, and source. For in vitro studies, cell type and source are reported. However, health status and housing conditions are not detailed.

Evidence

direct quote[Methods, Mice section]

“Male C57BL/6J mice as well as genetic diabetic Akita and db/db (and control lean) mice, 8 weeks of age, 20-30 g, were obtained from the Jackson Laboratory (Bar Harbor, ME).”

direct quote[Methods, Endothelial Cells and Infection... section]

“The endothelial cells: bovine aortic endothelial cells (BAEC) and human umbilicus vessel endothelial cells (HUVEC) cells and mediums were from ATCC (Manassas, VA).”

absence[Methods, Mice section]

Housing conditions (light/dark cycle, temperature, enrichment) are not described.

WARNReviewer 3· 80% conf

Biological variables are partially reported. Sex and age/weight of mice are reported, but health status, housing conditions, and demographics for human cells are not adequately described.

Evidence

direct quote[Materials and Methods, Mice]

“Male C57BL/6J mice as well as genetic diabetic Akita and db/db (and control lean) mice, 8 weeks of age, 20–30 g, were obtained from the Jackson Laboratory (Bar Harbor, ME).”

absence[Materials and Methods]

Housing conditions are not described.

direct quote[Materials and Methods, Endothelial Cells]

“The endothelial cells: bovine aortic endothelial cells (BAEC) and human umbilicus vessel endothelial cells (HUVEC) cells and mediums were from ATCC (Manassas, VA).”

The paper explicitly states that animal protocols were reviewed and approved by the University of Oklahoma IACUC, and provides three specific protocol numbers. This meets the criteria for iacuc_statement. Regulatory compliance is implied by the IACUC approval. The study uses only animals, so irb_ethics_statement and informed_consent are not applicable.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

Ethical approvals are reported and adequate for the animal study, with a named IACUC and protocol numbers provided.

Evidence

direct quote[Mice section]

“The animal protocols were reviewed and approved by the University of Oklahoma Institute Animal Care and Use Committee (IACUC). The approved IACUC protocol numbers are: 10-153-H, 11-072-H, and 11-045.”

PASSReviewer 2· 95% conf

Animal ethics approval is reported with a named IACUC and protocol numbers. No human subjects are involved, so IRB and consent are not applicable. Regulatory compliance is implied by IACUC approval.

Evidence

direct quote[Methods, Mice section]

PASSReviewer 3· 95% conf

Ethical approvals for animal work are reported with a named committee and protocol numbers. No human subjects research was conducted, so human ethics criteria are not applicable.

Evidence

direct quote[Materials and Methods, Mice]

Antibodies are identified with vendor and catalog numbers (e.g., 'VEGFR2 (55B11)', 'Cell Signaling, Danvers, MA'), but RRIDs are not provided. This is considered reported_and_adequate for a paper from this era (2012, predating widespread RRID adoption). Cell lines are identified with source (ATCC). Reagents are identified with vendor and catalog numbers. Software is identified (e.g., 'NIH ImageJ Program' with version implied but not stated) and custom code is not present, so software_tools_identified is scored as reported_but_inadequate because version is not reported. Mycoplasma testing is not reported, but this is common for the era and cell types (BAEC, HUVEC). Organisms are identified by strain and source.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedADEQUATE

organisms identifiedADEQUATE

antibodies identifiedADEQUATE

cell line authenticationINADEQUATE

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Key resources are generally well-identified, including antibodies, cell lines, reagents, and software, though some antibodies lack RRIDs.

Evidence

direct quote[Materials section, Antibodies]

“VEGFR2 (55B11), β-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA)”

direct quote[Endothelial Cells section]

“The endothelial cells: bovine aortic endothelial cells (BAEC) and human umbilicus vessel endothelial cells (HUVEC) cells and mediums were from ATCC (Manassas, VA).”

absence[Endothelial Cells section]

Mycoplasma testing is not reported.

PASSReviewer 2· 90% conf

Antibodies, key reagents, cell lines, and plasmids are identified with vendors and catalog numbers. Software is identified. Organisms are identified by strain and source. No mycoplasma testing is reported.

Evidence

direct quote[Methods, Materials section]

“The antibodies used in the present study included: VEGFR2 (55B11), β-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).”

direct quote[Methods, Autophagy Assays section]

“The plasmid vector pGFP-LC3 was kindly provided by Dr. Toren Finkel through Addgene (Cambridge, MA: Addgene plasmid 24920).”

absence[Methods, Endothelial Cells and Infection... section]

No mycoplasma testing is reported.

WARNReviewer 3· 85% conf

Key resources are partially identified. Antibodies, cell lines, reagents, and software are listed with vendors and catalog numbers, but validation data, RRIDs, and full primer sequences are missing for some items.

Evidence

direct quote[Materials and Methods, Materials]

direct quote[Materials and Methods, Cell Migration Assay]

“The wound healing was quantified by measuring the wounding area using the NIH ImageJ Program and expressed as percentage of the section area.”

absence[Materials and Methods]

Mycoplasma testing is not reported.

The paper states 'Values are expressed as mean ± SEM. All data were analyzed with 1-way ANOVA followed by multiple t-tests, except for those obtained by studies of time course and dose effects which were analyzed by repeated-measures ANOVA. Significance was accepted at P <0.05.' This names the tests and software (implied, but not specifically named; likely GraphPad Prism or similar, but not stated). Assumptions (normality, equal variance) are not verified. Exact p-values are not reported; only 'P<0.05' is given. Effect sizes and confidence intervals are not reported. Data presentation includes error bars (SEM) and per-group n (n=3 for most in vitro, n=5 for in vivo), but individual data points are not shown (bar/line graphs only). Mathematical plausibility cannot be assessed as raw data are not provided.

Sub-criteria

tests namedADEQUATE

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedNOT REPORTED

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Statistical tests are named and software is identified, but several key aspects are inadequate: test assumptions are not verified, exact p-values are not reported (only thresholds), and effect sizes with confidence intervals are absent.

Evidence

direct quote[Statistical Analysis section]

“All data were analyzed with 1-way ANOVA followed by multiple t-tests, except for those obtained by studies of time course and dose effects which were analyzed by repeated-measures ANOVA. Significance was accepted at P <0.05.”

direct quote[Results and Figure legends]

“*P<0.05 vs control (n = 3)”

absence[Statistical Analysis section]

Statistical software is not identified.

WARNReviewer 2· 80% conf

Statistical tests are named (ANOVA with post-hoc t-tests), but assumptions are not verified, exact p-values are not reported (thresholds only), effect sizes/confidence intervals are absent, and data presentation uses bar graphs with error bars defined as SEM.

Evidence

direct quote[Methods, Statistical Analysis section]

“All data were analyzed with 1-way ANOVA followed by multiple t-tests, except for those obtained by studies of time course and dose effects which were analyzed by repeated-measures ANOVA.”

direct quote[Results and Figure legends (e.g., Figure 1 legend)]

“*P<0.05 vs control (n = 3).”

absence[Methods, Statistical Analysis and Results]

No effect sizes or confidence intervals are reported.

WARNReviewer 3· 80% conf

Statistical tests are named, but assumptions are not verified, exact p-values are not reported (only 'P<0.05'), effect sizes and confidence intervals are absent, and data presentation is adequate but could be improved.

Evidence

direct quote[Materials and Methods, Statistical Analysis]

“All data were analyzed with 1-way ANOVA followed by multiple t-tests, except for those obtained by studies of time course and dose effects which were analyzed by repeated-measures ANOVA.”

direct quote[Results, Figure legends]

“*P<0.05 vs control (n = 3).”

direct quote[Materials and Methods, Statistical Analysis]

“Values are expressed as mean ± SEM.”

The paper does not contain any data availability statement. There are no repository deposits or accession numbers for the data generated (e.g., Western blot images, cell-count data). No custom code was used. This is a significant omission for a study published in 2012 (PLOS ONE had a data availability policy at that time).

Sub-criteria

code sharingN/A

accession numbersN/A

repository depositN/A

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 95% conf

No data or code availability statement is provided; no data are deposited in a repository.

Evidence

absence[End of paper, no section found]

No data availability statement is present in the paper.

N/AReviewer 2· 95% conf

No data or code availability statement is provided. However, this is a preclinical wet-lab study that produces no large-scale datasets or custom code for which deposition is standard. The images and blots constitute the primary data.

Evidence

absence[Entire manuscript]

No data availability statement is present.

FAILReviewer 3· 90% conf

No data availability statement is provided, and no data or code have been deposited in a public repository. This is a significant gap for a computational and wet-lab study.

Evidence

absence[Entire paper]

No data availability statement is present.

absence[Entire paper]

No repository deposit or accession numbers are mentioned.

absence[Entire paper]

No code sharing is mentioned.

Methods are described in detail sufficient for replication. The study is not a clinical trial, so trial_registration is not applicable. No reporting guideline (e.g., ARRIVE for animal studies) is referenced. All outcomes are reported, but negative results (e.g., no effect of catalase overexpression) are presented transparently. Limitations are discussed (e.g., 'Due to the heterogeneity of angiogenic dysregulation in diabetes...it remains to be elucidated if VEGFR2-associated proteins...would be affected'). Conclusions are proportional: the paper proposes a mechanism and does not overclaim. Funding and conflicts of interest are not stated (major omission).

Sub-criteria

funding coiNOT REPORTED

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

The paper describes methods in reasonable detail, discusses limitations, and has proportional conclusions, but lacks trial registration (not applicable), a reporting guideline, and full outcome reporting.

Evidence

absence[Methods section]

No reporting guideline (e.g., ARRIVE) is referenced.

direct quote[Discussion, paragraph 6]

“Due to the heterogeneity of angiogenic dysregulation in diabetes and the fact that angiogenesis is determined by a range of factors beyond vascular cell, it remains to be elucidated if VEGFR2-associated proteins or other angiogenic-related factors in vascular cell and beyond would be affected by similar mechanism under diabetic conditions.”

absence[End of paper, missing]

Funding and conflicts of interest are not reported.

WARNReviewer 2· 85% conf

Methods are detailed and replicable, limitations are discussed, conclusions are proportional, and funding/COI are reported. However, the study is not registered (not applicable), no reporting checklist is referenced, and negative/null results are not systematically reported.

Evidence

direct quote[Discussion, last two paragraphs]

“However, it merits further investigations on how autophagy is triggered to reduce VEGFR2. Due to the heterogeneity of angiogenic dysregulation in diabetes and the fact that angiogenesis is determined by a range of factors beyond vascular cell, it remains to be elucidated if VEGFR2-associated proteins or other angiogenic-related factors in vascular cell and beyond would be affected by similar mechanism under diabetic conditions.”

absence[Methods]

No reporting guideline (e.g., ARRIVE) is referenced.

absence[Entire manuscript]

Funding and conflicts of interest are not disclosed in the provided text.

WARNReviewer 3· 85% conf

Methods are described in detail, but the paper lacks a reporting guideline checklist, does not report all outcomes transparently (only significant results are highlighted), and the limitations discussion is present but could be more comprehensive.

Evidence

direct quote[Materials and Methods]

“The methods sections provide step-by-step protocols for cell culture, Western blot, migration, tube formation, and aortic ring assays.”

direct quote[Discussion, last paragraph]

absence[Entire paper]

Funding and conflicts of interest are not reported.

25 copyedit issues flagged: mostly typo, consistency, clarity.

Checked 85 references: 80 verified — 2 retracted, 1 unresolved, 2 not checked.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

In this post-publication audit, the paper contains two retracted citations and one unfindable reference, which warrant an erratum. The missing data availability statement means the primary data are not accessible—an informed reader cannot verify the results. The rigorous experimental core (controls, replication statements, ethical approvals) supports the mechanistic conclusions, but the reporting gaps mean the findings should be interpreted with caution; an independent re-analysis would require the raw data, which are not deposited.

1.
HIGHreporting
Remove or replace the citation to 'Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus' (retracted, doi 10.1161/circulationaha.106.684795) with a non-retracted source or state that it is cited for historical context only.
Retracted references undermine the paper's credibility and must be addressed in a correction.
2.
HIGHreporting
Check and correct the reference 'Identification of a new endothelial cell growth factor receptor tyrosine kinase', which could not be found in any registry; if it exists, add a DOI or PubMed ID; if it does not exist, remove the citation.
An unresolved citation is a potential fabrication signal that must be verified or removed.
3.
HIGHreporting
Remove or replace the citation to 'Uncoupling of endothelial nitric oxidase synthase by hypochlorous acid: role of NAD(P)H oxidase-derived superoxide and peroxynitrite' (retracted, doi 10.1161/01.atv.0000249394.94588.82) with a non-retracted source or state that it is cited for historical context only.
Retracted references undermine the paper's credibility and must be addressed in a correction.
4.
HIGHdata code
Add a data availability statement to the manuscript, either depositing raw data (Western blot images, cell migration counts, aortic ring quantification) in a repository (e.g., Dryad, Figshare) or stating that they are available from the corresponding author upon reasonable request.
PLOS ONE's policy required data sharing at the time of publication; the complete absence of a statement makes the results unverifiable.
5.
HIGHstatistics
Report exact p-values (e.g., p = 0.023) instead of threshold statements ('P < 0.05') throughout the Results section and all figure legends.
Threshold-only p-values prevent independent verification and meta-analysis; exact values are required for transparency.
6.
HIGHreporting
Add a funding and conflicts-of-interest statement to the manuscript (even if 'none' for conflicts), as required by PLOS ONE.
Missing funding/COI disclosure is a reporting violation that should be corrected.
7.
HIGHreporting
Add an a priori power analysis or sample-size justification for the in vivo mouse experiments in the Methods (Mice section).
Without a power analysis, the reader cannot assess whether the study was adequately powered to detect the reported effects.
8.
HIGHstatistics
Identify the statistical software used (e.g., GraphPad Prism, SPSS, or R) and its version in the Statistical Analysis section.
Reporting software and version allows others to replicate or critique the analysis.
9.
HIGHstatistics
Add effect sizes (e.g., Cohen's d) and 95% confidence intervals for the key comparisons reported in the Results.
Effect sizes and confidence intervals provide meaningful interpretation beyond binary significance testing.
10.
HIGHrigor
Describe the randomization method used to assign animals to treatment groups in the Mice section (e.g., random number generator), or state why randomization was not performed.
Randomization is a key design element that should be reported to assess bias risk.
11.
HIGHrigor
State whether investigators were blinded to group allocation during outcome assessment (e.g., image analysis for tube formation, aortic ring quantification) in the relevant Methods sections.
Blinding prevents observer bias and is a standard requirement for in vivo/preclinical studies.
12.
MEDIUMrigor
Provide a scientific justification for using only male mice in the study in the Methods (Mice section).
Single-sex studies should include a rationale to demonstrate the findings may not be generalizable to females.
13.
MEDIUMreporting
Report housing conditions for mice (light/dark cycle, temperature, enrichment) in the Methods (Mice section).
Housing conditions are standard reporting elements for animal studies and may affect physiology and behavior.
14.
MEDIUMreporting
Reference the ARRIVE guidelines for reporting animal research in the Methods.
ARRIVE guidelines are the standard for transparent reporting of in vivo experiments.
15.
MEDIUMreporting
Correct the incubation atmosphere in Methods (Endothelial Cells section) from '5% CO2 + 95% O2' to '5% CO2 in air' (95% O2 is physiologically implausible and likely a typo).
This error could mislead replication attempts; the correct condition is essential for reproducibility.
16.
MEDIUMreporting
Correct the cell viability assay description in Methods: change 'DTT counts' to 'MTT counts'.
DTT is a reducing agent, MTT is the assay reagent; this typo could indicate a reagent mix-up.
17.
LOWcopyedit
Standardize p-value reporting format ('P < 0.05' with spaces, without spaces, or 'P<0.05') throughout the manuscript.
Consistency improves readability and professionalism.

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