1. Evaluation Summary:

    The authors developed an aorta-on-a-chip system to investigate the potential mechanism of Bicuspid Aortic Valve-Thoracic Aortic Aneurysm, BAV-TAA, and to test candidate pharmacologic agents. This study is of broad interest to readers in the field of lab-on-a-chip, biomedicine, biomaterials, etc. However, some important details regarding the development of the model are missing, as are some control data. The authors need to discuss the limitations of this model, such as the inability of their on-a-chip model to recapitulate aortic changes associated with complex pathologic processes such as ECM degradation, inflammation, etc, and discuss the importance of follow-up studies in in vivo models.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    The authors developed an effective aorta-on-a-chip system to investigate the potential mechanism of Bicuspid Aortic Valve-Thoracic Aortic Aneurysm, BAV-TAA. They found that the impaired mitochondrial dynamics were involved in the development of BAV-TAA. Also, the drug screening tests involving mitochondrial fusion activators or mitochondrial fission inhibitor, which could rescue the dysfunctional VSMCs, were conducted on this system. These results could provide us an insight that this microphysiological system could serve as the potentially effective research platform for BAV-TAA. The physiologic mimicry of the aortaon-a-chip proved to be excellent and was superior to the 2D aortic cell research-in-a-dish. However, there are still some minor issues that need to be addressed.

    1. The authors used aortic smooth muscle cell as a basis for their chips. Although the aortic smooth muscle cells and its related tunica media play a central role in the development of aortopathy, other multiple types of cells, including endothelial cells, fibroblasts and macrophages, are also involved in the aortic wall and contribute to the pathological process. Indeed, this work could inspire future work of integration of other cell types on the chip to study their network effect involved in the aortopathy. However, the present study cannot represent the whole aortic wall on the chip. Therefore, the authors should rephrase the statement of 'aorta-on-a-chip' in the entire manuscript since the current wording leaves the impression that they integrate entire aortic cells. The "aorta tunica media-on-a-chip" could be more precise for the topic.

    2. The authors used primary smooth muscle cells form patients with BAV-TAA. However, the population of VSMCs comprising the aortic microstructures is consequently heterogeneous. In the ascending aorta and aortic arch, VSMCs are derived from the neural crest; VSMCs in the descending thoracic aorta are derived from the paraxial mesoderm, while neural crest and secondary heart field-derived VSMCs intermingle in the aortic root. The heterogeneity of VSMCs could lead to section-specific microphysiology in the aortic wall and differences in the vulnerability of VSMCs to pathogenic stimuli. Therefore, the authors should clarify which section of aorta they used and explain why they used that section.

    3. Although this is the first report of aortic function unit-on-a-chip to study aortopathy, the authors should clarify in more detail what discriminates this study from the other reported 'artery-on-a-chip' platforms (Artery-on-a-chip platform for automated, multimodal assessment of cerebral blood vessel structure and function, Lab Chip 2015;15(12):2660-9.doi: 10.1039/c5lc00021a) and what the level of novelty is.

    4. In Figure 6, the patients-derived cells were used on the chip. The effect of drug testing on aorta-on-a-chip model was desirable, even though the results did not exhibit all the positive responses. The authors should explain the potential reasons for the unevenness.

    5. The authors should explain why the commercial primary cells and primary isolated cells should be used together.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Abudupataer et al. have picked an important scientific/clinical problem and have made some interesting discoveries. They described a microphysiological device for the purpose of building an in vitro model of the aortic aneurysm. After design validation, they used the model to study the role of impaired mitochondrial dynamics and tested drugs to implicate the potential therapeutic targets. Impaired mitochondrial dynamics has already been linked to several cardiovascular conditions including ischemia-reperfusion injury and pulmonary arterial hypertension; and this study provides in vitro data suggesting its role in bicuspid aortopathy-related aortic aneurysm. This manuscript has considerable merit to the field.

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  4. Reviewer #3 (Public Review):

    Abudupataer et al. investigated the association between mitochondrial dynamics and NOTCH1 deficiency in BAV-TAA using an engineered aorta-on-a-chip model. They found that NOTCH1 insufficiency induced phenotypic switching in human aortic smooth muscle cells (HAoSMCs) and that MFN1/2 agonists and DRP1 inhibitors could be a potential therapeutic approach to reverse the imbalance in mitochondrial dynamics. The authors performed systematic in vitro evaluation of mitochondrial function and dynamics in HAoSMCs. The main conclusions of this paper are supported well by the data provided. However, it can be improved by addressing the following questions.

    1. Although microfluidics-based organ-on-a-chips have provided tremendous benefits in the biomedical researches, it is still premature to establish this method as a standard preclinical model. Thus, it is strongly recommended that the authors can conduct in vivo validation using animal model to explore the outcomes of leflunomide, Mdivi-1, and teriflunomide in the treatment of BAV-TAA.

    2. It would be more convincing if the authors provide sound justification regarding the strain of the ascending aorta, including its definition, the common value range, and its correlation with vacuum pressure, in the section of "Construction of aorta-on-a-chip model" rather than "Discussion".

    3. In Figure 1f, the z score of each identified signaling pathway should be provided.

    4. In the horizontal axis of Figure 1h, is it log2 or log10? Please specify.

    5. How about the stability or repeatability of the chip for a long time? Please discuss and provide supporting evidence.

    6. What the implication of the enhancement of cell contractility? Further discussion is needed.

    7. Could the authors give a further explanation why there is a decrease in long rod-shaped of mitochondrial for NOTHC1-KD HAoSMCs?

    8. What's the criteria to decide upon the right vacuum pressure? Will the different tensile strain greatly influence the performance of the chip? How about the shape of the HAoSMCs under strain in the chip compared to the real sample?

    9. Could the author describe in greater details the major advantages of the aorta-on-a-chip model proposed in this manuscript compared to the conventional cell culture model?

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