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: 58
Author: Αlpha¹

Paper source

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: moderateReviewer agreement: lowEngine: 7.5.0Source: full text

Not verified in this report

References were not verified against Crossref/OpenAlex.
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 establishes a clear scientific premise and reports adequate ethical approvals, but suffers from major gaps in study design rigor (no randomization, blinding, power analysis), incomplete statistical reporting (no exact p-values, effect sizes, post-hoc specification), and absent data/code availability. Biological variables and key resources are partially reported but missing key details.

Two independent reviewers diverged on study design, biological variables, key resources, statistical analysis, and reporting transparency; the synthesized statuses were determined by checklist-level arithmetic and weighted evidence, with the more conservative (fail) status adopted for study design and statistical analysis due to concrete missing elements. The statistics verification component found no testable statistics (0 tests recomputed), so no errors were detected, but the absence of reporting is itself a failing. The paper is from 2012, which may explain some omissions but does not alter the rigor assessment.

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

Rigor

Eight Dimensions2 pass · 2 warn · 4 fail

11 major claims1 partial10 supported

Prior work on VEGFR2 reduction in diabetes and MGO's role in diabetic complications is cited. The rationale that MGO may reduce VEGFR2 via autophagy is logically developed. Limitations of prior research are acknowledged, such as the incomplete understanding of diabetic angiogenesis impairment.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 90% conf

Strong scientific premise clearly established. The study builds on extensive prior work documenting VEGFR2's role in angiogenesis, MGO's role in diabetes, and the gap in knowledge linking these.

Evidence

direct quote[Introduction, paragraphs 1–3]

“Methylglyoxal (MGO) is the major source of intracellular advanced glycation end-products (AGEs) ... MGO has been implicated in the pathogenesis of diabetic complications.”

direct quote[Introduction, final paragraph]

“the mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known... this study investigated the impacts of MGO on VEGFR2 protein levels and endothelial angiogenesis”

paraphrase[Introduction, paragraphs 1 and 4]

“The introduction states multiple mechanisms have been proposed but diabetes-impaired angiogenesis is not completely understood.”

PASSReviewer 2· 95% conf

The paper establishes a clear scientific premise by citing prior work on VEGFR2, MGO, and diabetes, and logically links the hypothesis to gaps in knowledge.

Evidence

direct quote[Introduction]

“VEGFR2 protein reduction has been observed in patients with diabetes, and in experimental diabetic animals. The mechanism underlying VEGFR2 reduction and the contributions to angiogenesis impairment in diabetes are not known.”

direct quote[Introduction]

“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...”

direct quote[Introduction]

“However, it has not been completely elucidated how diabetes impairs physiological angiogenesis.”

Randomization, blinding, and power analysis are not reported. Replicate distinction is unclear (n=3 but not defined as biological vs technical). Inclusion/exclusion criteria and outlier handling are absent. Controls are adequate. Independent replication is not reported.

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 (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Core experimental rigor is moderate. Key elements like randomization, blinding, and power analysis are not reported for the in vivo mouse experiments. Replicate distinction is clear for cell work, but outlier handling and independent replication are not explicit.

Evidence

direct quote[Figure legends and Methods]

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

absence[Materials and Methods]

Randomization, blinding, and power analysis are not mentioned anywhere in the manuscript.

paraphrase[Results, Figures 1 and 8]

“MGO reduces VEGFR2 in BAEC and HUVEC (data not shown). Aortic angiogenesis is impaired in both db/db (Fig 8B) and Akita (Fig 8C) mice.”

FAILReviewer 2· 90% conf

The study lacks randomization, blinding, power analysis, and many other design rigor elements.

Evidence

absence[Methods]

No randomization method reported

absence[Methods]

Blinding not mentioned

paraphrase[Methods, Results]

“The paper states 'All blots shown are representative of three independent experiments' but does not explicitly define whether these are biological or technical replicates.”

For mice: sex is reported (male), age (8 weeks) and weight (20-30 g). Strain (C57BL/6J) and source (Jackson Laboratory) are given. No scientific justification is provided for using only male mice (cost/convenience would be inadequate). Housing conditions (light cycle, temperature, etc.) are not described. For cell lines: BAEC and HUVEC are identified with source (ATCC), but passage number is not reported or only partially ('used between passages 3-8'). No health status or sex is reported for cell lines (n/a for primary cells). Species is reported (bovine, human). Demographics (for human HUVEC) are not applicable as the source is commercial. Per scoring rules for preclinical interventional studies, 5 sub-criteria apply. Sex, age/weight, species/strain are adequate; sex_justification and housing are missing.

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedNOT REPORTED

age weight healthADEQUATE

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 90% conf

Biological variables are partially reported. Sex, age, and weight of mice are stated, but sex justification and housing conditions are missing. Cell line source is reported, but passage number information is incomplete.

Evidence

direct quote[Materials and Methods, Mice paragraph]

“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, Mice paragraph]

No scientific justification for using only male mice is provided.

absence[Materials and Methods]

Housing/husbandry conditions are not described.

PASSReviewer 2· 85% conf

Sex, age, weight, strain, and source are reported for mice, but housing conditions are not described.

Evidence

direct quote[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”

absence[Methods]

Housing conditions not reported

absence[Methods]

No justification for single-sex

The paper reports that animal protocols were reviewed and approved by the University of Oklahoma Institute Animal Care and Use Committee (IACUC), with three specific protocol numbers provided. This meets the standard for 'reported_and_adequate'. No human participants were involved, so IRB/consent criteria are not applicable. The study uses commercial cell lines (ATCC) which do not require ethics approval.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceINADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 95% conf

Animal ethics approval is adequately reported with a named committee and protocol numbers. No human subject research was conducted.

Evidence

direct quote[Materials and Methods, Mice paragraph]

“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.”

absence[Materials and Methods]

Regulatory compliance (e.g., Guide for the Care and Use of Laboratory Animals) is not explicitly stated.

PASSReviewer 2· 95% conf

Animal ethics approval is reported with IACUC protocol numbers, meeting the standard for animal research.

Evidence

direct quote[Methods]

Antibodies are listed with vendor and clone but not catalog numbers. Cell lines are from ATCC but no authentication or mycoplasma testing reported. Organisms are adequately identified. Software tools are not identified by version. The majority of applicable criteria are inadequate.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedINADEQUATE

organisms identifiedADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationINADEQUATE

software tools identifiedNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Key resources are identified with vendors and catalog numbers, but several deficiencies exist: antibodies lack RRIDs, cell lines lack STR authentication, mycoplasma testing is not reported, and primer sequences are not fully written in the standard orientation.

Evidence

direct quote[Materials and Methods, Materials paragraph]

“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)... (Santa Cruz Biotechnology, Santa Cruz, CA)”

absence[Materials and Methods, Endothelial Cells paragraph]

Cell line authentication and mycoplasma testing are not mentioned.

direct quote[Materials and Methods, RT-PCR paragraph]

“Forward and reverse specific primer sequences were: VEGFR2 , forward: 5′-TGTGGGTTTGCCTAGTGTTTCT ; reverse: 5′-CACTCAGTCACCTCCACCCTT”

FAILReviewer 2· 85% conf

Antibodies, reagents, and cell lines are identified by vendor but lack catalog numbers, RRIDs, and authentication details.

Evidence

direct quote[Methods]

“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]

“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]

Statistical software not identified

Tests named (ANOVA, t-tests) but post-hoc method not specified. Assumptions not verified. P-values reported as thresholds only. No effect sizes or confidence intervals. Statistical software not identified. Data presentation uses bar graphs without individual points.

Sub-criteria

tests namedINADEQUATE

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedNOT REPORTED

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

Statistical analysis is adequate for the study type, with tests named, exact p-values reported in graphs, and software implied (but not stated). However, test assumptions are not verified, effect sizes with CIs are absent, and the main statistical method (multiple t-tests after ANOVA) is problematic for multiple comparisons.

Evidence

direct quote[Materials and Methods, Statistical Analysis paragraph]

“All data were analyzed with 1-way ANOVA followed by multiple t-tests... Significance was accepted at P <0.05.”

direct quote[Results and Figure Legends]

“*P<0.05 vs control (n = 3).”

absence[Results and Statistical Analysis]

Effect sizes and confidence intervals are not reported.

FAILReviewer 2· 90% conf

Statistical tests are named but not fully specified, assumptions are not verified, exact p-values are not reported, and individual data points are not shown.

Evidence

direct quote[Methods]

“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]

“*P<0.05 vs control (n = 3).”

paraphrase[Figures]

“Figures display bar graphs with error bars, not individual data points.”

The paper does not include a data availability statement, and no data or code repositories are referenced. This is a significant omission for a modern research paper, though the paper is from 2012.

Sub-criteria

code sharingNOT REPORTED

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 95% conf

No data availability statement, repository deposit, or code sharing is reported. This is a significant gap for a journal that typically requires data access statements.

Evidence

absence[Entire manuscript]

No data availability statement exists in the paper.

absence[Entire manuscript]

No repository deposit or accession numbers are mentioned.

absence[Entire manuscript]

No code sharing statement is present; custom analysis methods are described textually but not with provided code.

FAILReviewer 2· 95% conf

No data availability statement, data deposition, or code sharing is provided.

Evidence

absence[Entire paper]

No data availability statement

absence[Entire paper]

No repository deposit

absence[Entire paper]

No code sharing

Methods are detailed and include specific protocols, catalog numbers, and conditions, making replication feasible. No reporting guideline (e.g., ARRIVE for animal studies) is referenced. Limitations are briefly discussed in the final Discussion paragraph, but the coverage is superficial and does not address key issues like the lack of randomization/blinding or the use of only male mice. Conclusions are generally proportional to the evidence presented in this study, but the claim of a 'new mechanism' is somewhat overstated given the observational nature of most data. Funding sources and conflicts of interest are not reported (the manuscript appears to be from a pre-2013 era where this was not standard on the first page, but it remains a gap). Trial registration does not apply. 'All outcomes reported' is not verifiable without a pre-registration, but no outcome omissions are obvious; the study reports negative results (e.g., no change in VEGFR2 mRNA, no effect of catalase).

Sub-criteria

funding coiNOT REPORTED

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedINADEQUATE

conclusions proportionalADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Reporting completeness is moderate. Methods are detailed enough for replication, but key elements like limitations discussion are present but weak, and the paper lacks a reporting checklist reference.

Evidence

paraphrase[Materials and Methods]

“The methods section describes cell culture, reagents, Western blot, cell viability, migration, tube formation, aortic ring assay, and RT-PCR in substantial detail.”

absence[Materials and Methods]

No reporting guideline (ARRIVE) is referenced for the animal studies.

paraphrase[Discussion, final paragraph]

“Due to the heterogeneity of angiogenic dysregulation in diabetes and the fact that angiogenesis is determined by a range of factors... it remains to be elucidated...”

PASSReviewer 2· 85% conf

Methods are detailed, outcomes are reported, limitations are discussed, and conclusions are proportional, but no reporting guideline or funding/COI statement is provided.

Evidence

paraphrase[Methods]

“The Methods section includes detailed protocols for cell culture, Western blot, autophagy assays, aortic ring assay, etc.”

direct quote[Discussion]

“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]

No funding/COI statement

10 copyedit issues flagged (1 major): mostly clarity, typo, consistency.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

In post-publication audit, the paper's robustness is limited by the absence of randomization, blinding, power analysis, and full statistical reporting; an informed reader should weigh these gaps when interpreting the results. The authors should consider publishing a correction to address the missing reporting elements (e.g., data availability statement, exact p-values, post-hoc test specification) and to clarify the limitations. The missing data availability statement is a critical issue for reproducibility.

1.
HIGHdata code
Add a data availability statement and deposit raw data (e.g., Western blot images, aortic ring quantification, cell viability data) in a public repository like Dryad or Figshare, and include the repository link in the manuscript.
Without accessible data, the study's findings cannot be independently verified, undermining reproducibility.
2.
HIGHrigor
Report the method of randomization (e.g., computer-generated random numbers) and blinding procedures (who was blinded, at which stage) for animal treatments and outcome assessments in the Methods (Mice section, Aortic Ring Assay).
Randomization and blinding are essential to reduce allocation and assessment bias in animal studies; their absence weakens the validity of the conclusions.
3.
HIGHstatistics
Specify the post-hoc test used after ANOVA (e.g., Tukey, Bonferroni) and report exact p-values (e.g., p=0.023) instead of only thresholds in figure legends and text.
The current practice of 'multiple t-tests' without correction inflates Type I error, and threshold-only p-values prevent readers from assessing the strength of evidence.
4.
HIGHrigor
Report a priori power analysis or sample size justification for the in vivo mouse experiments in the Methods (Mice section).
Without power analysis, it is unclear whether the study had sufficient statistical power to detect the reported effects, raising concerns about false negatives/positives.
5.
HIGHrigor
Clarify whether n=3 refers to biological or technical replicates for all cell-based experiments, and define the distinction explicitly in the Methods.
Ambiguity between biological and technical replicates affects the interpretation of statistical analyses and the generalizability of results.
6.
HIGHrigor
Report cell line authentication (e.g., STR profiling) and mycoplasma testing results for BAEC and HUVEC in the Methods (Endothelial Cells section).
Unauthenticated or contaminated cell lines can produce misleading results; this is a recognized reproducibility issue.
7.
HIGHreporting
Provide a scientific justification for using only male mice in the animal experiments, addressing potential sex-specific effects, in the Methods (Mice section) or Discussion.
Single-sex studies without justification limit the generalizability of findings to female populations and may mask sex-dependent biology.
8.
HIGHreporting
Include a statement of funding sources and conflicts of interest at the end of the manuscript.
Transparency about funding and competing interests is a standard reporting requirement for all journals.
9.
HIGHreporting
Revise the title to a single, clear phrase (e.g., 'Role of Methylglyoxal-Induced Autophagy in VEGFR2 Degradation and Impaired Angiogenesis in Diabetes') to replace the current concatenated title.
The current title appears to be a concatenation of two separate phrases, which is confusing and unprofessional.
10.
MEDIUMrigor
Provide catalog numbers, RRIDs, and validation data (e.g., species reactivity, application) for all antibodies in the Methods (Materials section).
Antibody identification with RRIDs and catalog numbers is essential for reproducibility of Western blot and immunostaining experiments.
11.
MEDIUMreporting
Describe housing and husbandry conditions (light cycle, temperature, humidity, group size) for mice in the Methods (Mice section).
Housing conditions can affect physiological and behavioral outcomes; their absence limits reproducibility.
12.
MEDIUMstatistics
Show individual data points (e.g., dot plots) alongside bar graphs in figures where sample sizes are small (n=3-5).
Bar graphs with small sample sizes can obscure the distribution and variability of the data; individual points allow readers to assess outliers and data spread.
13.
MEDIUMstatistics
Identify the statistical software (e.g., GraphPad Prism, version) used for all analyses in the Methods (Statistical Analysis section).
Software version information is necessary for reproducibility of statistical computations.
14.
MEDIUMreporting
Include a completed reporting checklist (e.g., ARRIVE for animal studies) as a supplementary file.
Adherence to reporting guidelines improves transparency and completeness of methods reporting.
15.
LOWcopyedit
Fix grammatical and typographical errors: change 'took one step further' to 'took a step further' (Results, paragraph 7); change 'Mice Male' to 'Male' (Methods, Mice); change 'do not affect' to 'does not affect' (Results, paragraph 2); hyphenate 'vehicle-treated' (Figure 8 legend); rephrase the sentence in Discussion, paragraph 1 that begins with 'Because' to form a complete sentence.
Minor errors detract from the professionalism of the manuscript and can cause confusion.

Full text (66,286 chars)Analyzed: Jul 6, 2026

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