Review Reports - Mouse Models of Atherosclerosis: What They Teach Us, What They Miss, and When to Use Them

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Mouse models have played an indispensable role in studying the mechanisms of atherosclerosis, but no single model fully reproduces the spectrum of human disease. This review provides a practical basis for selecting mouse models based on the biological problem being addressed.

Atherosclerosis is a chronic immunometabolic disease characterized by the accumulation of lipids, inflammatory cells, smooth muscle cells, and necrotic debris in the arterial wall. Inflammation is recognized not as a secondary consequence of lipid accumulation, but as a fundamental driver of the disease. Mouse models remain indispensable, but no single model fully reproduces human pathology.
The authors analyzed a wide range of models:
1. Apoe⁻/⁻ mice: a classic model with accumulation of cholesterol-rich lipoprotein remnants. They develop spontaneous atherosclerosis even on a standard diet. However, ApoE has pleiotropic effects beyond lipoprotein clearance. 2. Ldlr⁻/⁻ mice: a complementary model requiring dietary intervention for atherosclerosis development. LDL/ApoB100 accumulation, ApoE preserved. Considered more representative of human LDL-mediated disease.
3. Immune and immunometabolic models: Ldlr⁻/⁻ is often preferred for immune studies due to its preserved ApoE. PCSK9-AAV and AAV-CRISPR Ldlr mice allow hypercholesterolemia to be induced in adult mice without germline modification. 4. Complex plaque and coronary disease models: Apoe⁻/⁻Fbn1C1039G⁻/⁻ (vulnerable plaques), SR-BI⁻/⁻Apoe⁻/⁻ (coronary disease and infarction), SR-BI⁻/⁻Ldlr⁻/⁻, AngII infusion models.
5. Human models: APOE3-Leiden, APOE3-Leiden.CETP, human ApoB and Lp(a) transgenic mice.

The authors emphasize the need for: precise model selection for the biological question; intermodel validation; improved reporting of experimental conditions; studying not only total cholesterol but also lipoprotein composition; integration with human data; and the use of adult induction systems.

1. Inclusion of key classic papers (Plump et al., 1992; Ishibashi et al., 1993)—fundamental articles on the creation of Apoe⁻/⁻ and Ldlr⁻/⁻ mice.
2. Inclusion of current references (2023-2026) on single-nucleus sequencing and new models.
3. Good coverage of methodological papers on diets and genetic background.

Some important references are missing:
No work on human atherosclerosis models in mice with "humanized" lipoproteins is mentioned, except for APOE3-Leiden.
Few references are provided on comparative studies of Apoe⁻/⁻ and Ldlr⁻/⁻ in different genetic backgrounds (only Torikai et al., 2023).

The article does not simply list models; it proposes a question-oriented selection strategy. This significantly increases the practical value for researchers.
The authors candidly discuss the limitations of each model, including:
1. Pleiotropic effects of ApoE (impact on immunity, inflammation, platelets)
2. Diet dependence and variability
3. Differences in lipoprotein profiles between models

Modern methods, including new approaches:
1. Single-nucleus RNA sequencing (scRNA-seq)
2. AAV-mediated PCSK9 delivery
3. Somatic genome editing with CRISPR/Cas9

Practical recommendations: Detailed recommendations for choosing a model for specific questions.

Weakness of the scientific article:
1. The article does not provide specific recommendations on the timing of experiments (how many weeks of Western diet for different models, when different plaque stages appear).
2. Add a table with typical time points:
Apoe⁻/⁻ on Chow: 12-16 weeks for early plaques, 24-40 weeks for complex plaques. Ldlr⁻/⁻ on Western: 8-12 weeks for early plaques, 16-24 weeks for complex plaques. PCSK9-AAV: 8-16 weeks after injection.

This article is a valuable resource for atherosclerosis researchers, offering a practical basis for model selection. Its critical analysis, attention to experimental context, and call for standardization make it useful for both novice and experienced scientists.
Upon revision, the article may be accepted for publication.

Author Response

We thank the reviewers for their careful evaluation of our manuscript and for their thoughtful and constructive recommendations. We appreciate their recognition of the breadth, timeliness, and practical value of our question-oriented framework for selecting mouse models of atherosclerosis. We have revised the manuscript extensively in response to the comments, as detailed below.

Reviewer 1

Reviewer comment:
“No work on human atherosclerosis models in mice with ‘humanized’ lipoproteins is mentioned, except for APOE3-Leiden.”

Response:
We acknowledge that the previous version did not provide sufficient coverage of mouse models incorporating human lipoprotein components. These models are important because they permit more direct investigation of human ApoB100-containing particles, lipoprotein(a), CETP-dependent lipid redistribution, and responses to lipid-modifying therapies. Therefore, we have substantially expanded the sections describing models of human-like lipoprotein metabolism. We now discuss Ldlr^−/−/Apobec1^−/−and ApoB100-only models, human ApoB-transgenic mice, human ApoB/apo(a) models that produce authentic Lp(a), and APOE3-Leiden.CETP mice. For each model, we describe its principal applications, translational advantages, and limitations, including diet dependence, variable hypercholesterolemia, incomplete reproduction of human apo(a) heterogeneity, and the persistence of species-specific differences in vascular anatomy, immune responses, and plaque complications.

Reviewer comment:
“Few references are provided on comparative studies of Apoe^−/− and Ldlr^−/− in different genetic backgrounds (only Torikai et al., 2023).”

Response:
We acknowledge that the influence of genetic background was not sufficiently supported by comparative studies in the previous version. Genetic background can substantially modify plasma lipoprotein profiles, lesion burden, plaque distribution, inflammatory responses, and vascular-site susceptibility. Therefore, we have expanded the subsection on genetic background and included studies examining Apoe^−/− mice on FVB/N, BALB/c, 129/SvEv, DBA/2J, C3H/HeJ, and C57BL/6 backgrounds, as well as comparative studies of Ldlr^−/− mice on C57BL/6, FVB/N, and C3H backgrounds. We now emphasize that the magnitude and anatomical distribution of atherosclerosis depend on interactions among the disrupted gene, genetic background, diet, sex, and vascular site.

Reviewer comment:
“The article does not provide specific recommendations on the timing of experiments (how many weeks of Western diet for different models, when different plaque stages appear). Add a table with typical time points: Apoe^−/− on Chow: 12–16 weeks for early plaques, 24–40 weeks for complex plaques. Ldlr^−/− on Western: 8–12 weeks for early plaques, 16–24 weeks for complex plaques. PCSK9-AAV: 8–16 weeks after injection.”

Response:
We acknowledge that experimental timing is a critical consideration when selecting a model to investigate lesion initiation, progression, or advanced plaque characteristics. Therefore, we have added Table 2, which summarizes representative experimental timelines for commonly used models. The table includes chow-fed Apoe^−/− mice, Western-type diet-fed Ldlr^−/− mice, and PCSK9-AAV-induced models, together with representative early- and complex-lesion windows, typical applications, and important experimental considerations. We also clarify that these timelines are practical starting points rather than fixed thresholds because lesion kinetics depend on genetic background, sex, age, diet composition, vascular site, vector serotype and dose, efficiency of LDLR depletion, microbiota, and the criteria used to define lesion stage.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript bioengineering-4472296

Title: Mouse Models of Atherosclerosis: What They Teach Us, What They Miss, and When to Use Them

Authors: Xiaoxiao Geng, Annalisse R. McKee, Manuel Rosa-Garrido, and Isidoro Cobo

Journal: Bioengineering (Peer Review Draft)

The We review the major mouse models used in atherosclerosis research, including classic lipoprotein-driven models (Apoe−/− and Ldlr−/− mice), models with human-like lipoprotein metabolism, immune and immunometabolic models, adult-onset inducible models (notably AAV-PCSK9), models of plaque complexity and models of coronary artery disease/myocardial infarction. The authors offer a pragmatic question-based framework for model selection, discuss the influence of genetic background, diet, sex, age and vascular site on lesion formation and, uniquely, address inconsistencies across single-cell RNA-sequencing (scRNA-seq) studies. The review concludes by calling for cross-model validation, improved reporting standards, and integration of spatial and human benchmarking approaches. This is a broad, timely and well-conceived synthesis that fills a real gap in the atherosclerosis literature. There are some things that need to be clarified or expanded upon in terms of experimental rigor and reproducibility as described below.

Comments to Authors:

- Add a Table 1 in the main text instead of Supplement:The Appendix Table 1 is explicitly introduced and defined in the manuscript and is referenced in section headings. But the actual formatted table body is not in the main text of this draft. Appendix Table 1 is a useful quick-reference tool for readers selecting models, so be sure to embed the full formatted table into the appendix before final publication.
Add a flow chart or a graphical abstract for decision making

The manuscript stresses the importance of model selection in relation to the biological question multiple times (Lines 26-29, 122-126, 644-647). This decision process would be much easier to read and apply in practice if it would be summarized in a graphical workflow.

Discuss sex as a biological variable in more detail in Discussion section-

- Sex differences are discussed in several sections (Lines 22, 283–285, 593–600, 660–661), but a dedicated subsection summarizing how sex influences lesion development, immune responses, and therapeutic outcomes would strengthen the review. Expand the sex differences section to include data on how estrogen affects lesion development in Apoe−/− and Ldlr−/− mice, and whether AAV-PCSK9 models have been validated in female mice.

Provide a brief comparison of mouse and human atherosclerosis

The manuscript does address species differences (Lines 94-100, 475-477), but a summary table or boxed section comparing major physiological differences (lipoprotein metabolism, plaque rupture, coronary anatomy, immune responses) would strengthen the translational perspective.

Expand future perspectives regarding emerging technologies

Spatial transcriptomics and proteomics are briefly mentioned in lines 635-642. The manuscript would be further strengthened by a short paragraph on the incorporation of AI-assisted pathology, spatial multi-omics and machine learning into model selection.

Improve consistency in terminology

Ensure consistent terminology for dietary interventions such as “Western diet,” “high-fat/high-cholesterol diet (HFD-C),” and related nomenclature throughout the manuscript (e.g. Lines 149–150, 272–273, 374–385, 402–404).

Lines 373- 397: AAV-PCSK9 section: Please provide the AAV serotype (usually AAV8), the dose range (e.g., 1–5 × 10^11 vg/mouse), and the route of administration (intravenous tail vein injection) that were used in the published studies. Also discuss validation of the model in female mice and in different genetic backgrounds.
Correcting Typos and Proofreading

- Line 79: "dependnet" → dependent

- line 162: "systematic consequences" should probably be systemic consequences

- Line 422: “If the question concern” → If the question concerns

- Line 433: "that that" → remove duplicated word

- Line 409: “underlaying mechanism” → underlying mechanism

- Line 529: should therefore regarded → should therefore be regarded

- Line 665: Clean up template placeholder text: "Please refer to the CRediT taxonomy for citation of term explanation."

- Line 668: Please delete the instructions in draft under Funding: 'Please add: This research was supported by...

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Good but the English could be improved to more clearly express the research.

Author Response

Reviewer 2

Reviewer comment 1:
“Add a Table 1 in the main text instead of Supplement. The Appendix Table 1 is explicitly introduced and defined in the manuscript and is referenced in section headings. But the actual formatted table body is not in the main text of this draft. Appendix Table 1 is a useful quick-reference tool for readers selecting models, so be sure to embed the full formatted table into the appendix before final publication.”

Response:
We acknowledge that the complete model-comparison table should be directly accessible to readers and consistently cited within the manuscript. Therefore, we have included the full formatted table in the main manuscript as Table 1 and revised the corresponding citations throughout the text. The table provides a comparative overview of the genetic or experimental manipulation, predominant lipoprotein abnormality, dietary dependence, lesion phenotype, principal strengths and limitations, and recommended applications of each model. Due to the size of the Table, we have decided to include both Tables as Figures in the manuscript and work with the editorial team to have them included as a whole page before publishing.

Reviewer comment 2:
“Add a flow chart or a graphical abstract for decision making. The manuscript stresses the importance of model selection in relation to the biological question multiple times. This decision process would be much easier to read and apply in practice if it would be summarized in a graphical workflow.”

Response:
We acknowledge that a visual workflow would make the question-oriented framework easier to interpret and apply. Therefore, we have added a graphical abstract entitled “Question-driven selection of mouse models of atherosclerosis.” The workflow guides readers toward models appropriate for studying core plaque initiation and progression; LDL/ApoB100-driven disease; remnant, CETP, and lipid-lowering pharmacology; immune and inflammatory mechanisms; aging and complex immunometabolic risk; or plaque vulnerability and coronary-event-like outcomes. It also highlights important context modifiers, including genetic background, diet, sex, age, vascular site, feeding duration, microbiome, and experimental endpoint.

Reviewer comment 3:
“Discuss sex as a biological variable in more detail in Discussion section. Sex differences are discussed in several sections, but a dedicated subsection summarizing how sex influences lesion development, immune responses, and therapeutic outcomes would strengthen the review. Expand the sex differences section to include data on how estrogen affects lesion development in Apoe−/− and Ldlr−/− mice, and whether AAV-PCSK9 models have been validated in female mice.”

Response:
We acknowledge that sex is a major biological and experimental variable whose effects depend on the model, age, diet, vascular site, and analytical endpoint. Therefore, we have added a dedicated subsection entitled “Sex as a biological variable in model selection and interpretation.” This subsection summarizes sex-dependent differences in lesion burden and plaque composition in Apoe−/− and Ldlr−/− mice, the protective effects of estrogen demonstrated by ovariectomy and estradiol-replacement studies, and the potential for sex-dependent immune and therapeutic responses. We also discuss the validation of AAV-PCSK9 models in female mice, sex-dependent differences in vector performance and hypercholesterolemia, and the need to report and analyze sex-specific outcomes independently.

Reviewer comment 4:
“Provide a brief comparison of mouse and human atherosclerosis. The manuscript does address species differences, but a summary table or boxed section comparing major physiological differences, including lipoprotein metabolism, plaque rupture, coronary anatomy, and immune responses, would strengthen the translational perspective.”

Response:
We acknowledge that a concise comparison would strengthen the translational framework of the review. Therefore, we have added Box 1, “Key differences between mouse and human atherosclerosis,” to the Introduction. The box compares lipoprotein metabolism, plaque distribution and clinical complications, disease kinetics, and immune and vascular responses. It also emphasizes that no mouse model reproduces the complete spectrum of human disease and that model selection and interpretation should be guided by the specific biological or clinical feature being studied.

Reviewer comment 5:
“Expand future perspectives regarding emerging technologies. Spatial transcriptomics and proteomics are briefly mentioned. The manuscript would be further strengthened by a short paragraph on the incorporation of AI-assisted pathology, spatial multi-omics and machine learning into model selection.”

Response:
We acknowledge that emerging spatial and computational technologies are increasingly important for assessing the translational relevance of experimental models. Therefore, we have expanded the Future Perspectives section to discuss AI-assisted pathology, spatial transcriptomics, spatial proteomics, multiplex imaging, lineage tracing, and machine-learning-based integration of single-cell, lipoprotein, immune, histopathological, and therapeutic-response data. We also emphasize the need for representative training datasets, external validation across strains, sexes, diets, vascular sites, and imaging platforms, transparent reporting, and continued expert oversight.

Reviewer comment 6:
“Improve consistency in terminology. Ensure consistent terminology for dietary interventions such as ‘Western diet,’ ‘high-fat/high-cholesterol diet (HFD-C),’ and related nomenclature throughout the manuscript.”

Response:
We acknowledge that inconsistent dietary terminology can complicate comparisons among experimental studies. Therefore, we have revised the manuscript to standardize the nomenclature used for dietary interventions. We now use Western-type diet for the commonly used high-fat, cholesterol-containing atherogenic diet and high-fat/high-cholesterol when referring to studies that specifically use or define that designation. We have also clarified that diets grouped under these general terms may differ substantially in cholesterol content, fat source, carbohydrate composition, and cholate content and that these details should be reported explicitly.

Reviewer comment 7:
“AAV-PCSK9 section: Please provide the AAV serotype (usually AAV8), the dose range (e.g., 1–5 × 10^11 vg/mouse), and the route of administration (intravenous tail vein injection) that were used in the published studies. Also discuss validation of the model in female mice and in different genetic backgrounds.”

Response:
We acknowledge that these methodological details are essential for reproducibility and for understanding differences in the efficiency and severity of disease induction. Therefore, we have expanded the AAV-PCSK9 section to specify that published studies commonly use the AAV8 serotype, doses in the approximate range of 1–5 × 10^11 vector genomes per mouse, and intravenous administration, typically by tail-vein injection. We also discuss the successful application of the model in female mice and across different genetic backgrounds, while emphasizing that vector dose, construct, sex, strain, diet, hepatic transduction efficiency, and the degree of LDLR depletion can influence plasma lipid levels, lesion kinetics, and plaque phenotype.

Reviewer comment 8:
“Correcting Typos and Proofreading: Line 79: ‘dependnet’ → dependent; line 162: ‘systematic consequences’ should probably be systemic consequences; Line 422: ‘If the question concern’ → If the question concerns; Line 433: ‘that that’ → remove duplicated word; Line 409: ‘underlaying mechanism’ → underlying mechanism; Line 529: ‘should therefore regarded’ → should therefore be regarded; Line 665: Clean up template placeholder text; Line 668: Please delete the instructions in draft under Funding.”

Response:
We acknowledge these typographical, grammatical, and template-related errors and thank the reviewer for identifying them. Therefore, we have corrected all the specified errors, removed duplicated wording and template instructions, revised the Author Contributions and Funding statements, and carefully proofread the complete manuscript for grammar, terminology, formatting, and consistency.

We thank both reviewers again for their thoughtful recommendations. These revisions have substantially improved the completeness, reproducibility, accessibility, and translational value of the manuscript.