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Brief summary of the study :

• The study investigates the differential effects of immunosuppressive drugs, sirolimus and tacrolimus, on endothelial cell function using iPSC-derived endothelial cells. The findings are significant as they provide mechanistic insights into vascular remodeling in transplant recipients, highlighting the varied impacts of these drugs. The study is well-structured, and the methodology appears robust. However, several areas could benefit from clarification and expansion for better contextual understanding and impact.

Major comments :

1. Experimental Design and Controls:

   • While the study appropriately uses iPSC-derived endothelial cells, the selection of patient control lines requires elaboration. Were there differences in the baseline characteristics of these lines that could influence outcomes?

   • The focus on specific drug concentrations (1 µM for tacrolimus and 50 nM for sirolimus) is noted, but it would be beneficial to include a dose-response analysis to validate the observed effects.

2. Mechanistic Insights:

   • The study identifies a significant decrease in nitric oxide production and endothelial migration with sirolimus treatment but does not delve deeply into the underlying molecular mechanisms. Including data on potential signaling pathways affected by these drugs would enhance the interpretation of results.

3. Statistical Robustness:

   • Some analyses, such as those involving nitric oxide production and LDL uptake, could benefit from a larger sample size to confirm reproducibility and generalizability of the findings.

4. Comparative Impact of Drugs:

   • While the results differentiate between the effects of sirolimus and tacrolimus, it would be helpful to compare these findings with additional immunosuppressive agents to contextualize their unique or shared impacts on endothelial function.

Minor comments:

Terminology and Clarity:

• The term "iPSC-EC" is used throughout the text but is only expanded once. Ensuring consistent definition in early sections (e.g., Abstract and Introduction) would improve readability.

Figures and Visuals:

• Figure 1 and Figure 2 are well-presented but could benefit from clearer annotations. For example, including statistical significance markers (e.g., p-values) directly in the figure panels would aid immediate interpretation.

Textual Improvements:

• Certain sections, such as the Discussion, include dense sentences that could be broken down for better readability (e.g., the paragraph discussing dyslipidemia and atherosclerosis mechanisms). Overall thorough correction of sentence construction and grammatical errors is required.

Comments on reporting :

Data Accessibility:

• While the data availability statement indicates access upon reasonable request, making anonymized data publicly available could further promote transparency and reproducibility.

• All raw data from ImageJ analysis and Seahorse assays should be included in supplementary materials.

Statistical Reporting:

• Detailed reporting on effect sizes alongside p-values would improve the assessment of biological relevance.

Suggestions for future studies

• Investigate the effects of these drugs on additional vascular cell types, such as vascular smooth muscle cells or fibroblasts, to develop a more comprehensive understanding of their impact on vascular remodeling.

• Extend the study to include in vivo models to corroborate the in vitro findings and bridge the gap to clinical applicability.

• Explore molecular targets or signaling pathways (e.g., mTOR or calcineurin pathways) involved in the observed effects to identify potential therapeutic interventions for minimizing vascular side effects.

Conflicts of interest of reviewers : None declared.

Competing interests

The authors declare that they have no competing interests.