PONE-D-19-16565

Hypoxia Induction in Cultured Pancreatic Islets Enhances Endothelial Cell Morphology and Survival while Maintaining Beta-cell Function

PLOS ONE

Dear Dr. Rocheleau,

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Feng Zhao

Academic Editor

PLOS ONE

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2. For ease of reproducibility we would recommend that you amend your methods section and provide further details of how the pancreatic islets were isolated from 10- to 12- week-old male C57BL6 mice.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors continued their previous study on how to maintain the pro- and anti-angiogenic balance to preserve vascular structures in the cultured pancreatic islets, which is critical for maintaining tissue viability and revascularization for transplantation for diabetes treatment. By using cultured islets collected from mice as the model, it is proposed that CoCl2 treatment, which is a hypoxia mimic, causes slower loss of ECs, while islet viability and insulin secretion functionality were not significantly affected. They also showed the correlation between the increased HIF-α/VEGF-A resulted from CoCl2 treatment and the increased ECs survival. And this effect can be augmented by using flowing culture. The topic is interesting and the flow-through of this manuscript is very clear. Before final publication it is suggested to do the following adjustment.

1. Some terms are not properly defined. For example, EC is mentioned in Intro for the first time lacks explanation. What is +ve control (In figure 1 caption)? Sometimes HIF1α was used; sometimes HIF-1α or HIF-1 was used. Please double check and be consistent.

2. Figure captions, especially for Fig 3, are too long and distracting. The data are already self-explanatory. There is no need to repeat the experimental conditions. Meanwhile, some essential info is missing. Please add explanation on different fluorescent colors either in the figure or in the captions.

3. This concern is related to the sentence in the discussion section “we manipulated the communication between beta-cells and ECs using the hypoxia mimetic CoCl2 “.

Since this study used isolated islet, which contains multiple cell type, can increased of H1F-1α and VEGF-A be attributed to other cells? Please explain why authors only credit the communication between beta-cell and ECs?

4. The ANOVA tests performed according to the presented results are not proper. ANOVA test tells whether differences between groups are statistically significant. Then post hoc tests (such as Tukey or LSD) should be performed to detect where the difference(s) are. The result should include the suggested grouping, not individually comparing with control, which could easily makes Type 1 error. Please report the proper result unless there is a specific reason for using t-test after ANOVA. Thus an alert should be given in the paper, too.

Reviewer #2: This manuscript confirmed that incubation isolated islets with hypoxia mimic cobalt chloride (CoCl2) induced endothelial cell morphology and cell survival. This hypoxia induction did not impair the beta-cell function based on the data on insulin secretion and beta-cell survival. Importantly, CoCl2 increased expression of HIF-1α and VEGF-A. The authors also showed that culturing islets in a microfluidic device combined with CoCl2 enhanced endothelial cell morphology and survival.

Some concerns:

1. In both “Introduction” and “Discussion” part, the authors stated “Previous studies attempting to maintain islet-ECs during culture have investigated exogenous growth factors and inhibitors of antiangiogenic factors, or overexpression of pro-angiogenic genes and silencing of anti-angiogenic genes”. More detailed and updated research information needs to be described and discussed.

2. In all the figures, the multiple significant differences between groups on a bar graph were marked not clear. The resolution of microscopy images can be improved.

3. In Figure 1, CoCl2 significantly increased HIF-1α expression (8-fold), but had no effect on insulin secretion. However, previous data (J Biol Chem 286: 12524–12532) reported that a constitutive activation of HIF-1α inhibit GSIS. This should be addressed.

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Hypoxia Induction in Cultured Pancreatic Islets Enhances Endothelial Cell Morphology and Survival while Maintaining Beta-cell Function

PONE-D-19-16565R1

Dear Dr. Rocheleau,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Feng Zhao

Academic Editor

PLOS ONE