PIEZO1 Promotes the Migration of Endothelial Cells via Enhancing CXCR4 Expression under Simulated Microgravity

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the in the re-submitted files.

2. Major comments:

Comments 1: Although a proteomics analysis (as shown in figure 3) revealed that the expression of CXCR4 protein decreased in the PIEZO1-knocked down endothelial cells, there are no detailed analyzed data on the mechanisms of the regulation of CXCR4 expression beyond this finding. Therefore, stating that 'we showed that upregulation of PIEZO1 induced by simulated microgravity increased CXCR4 protein level through Ca²⁺ influx (in lines 228-229) is an overstatement. A detailed analysis of the CXCR4 expression increase induced by microgravity is needed, using PIEZO1 knockdown, PIEZO1 agonists, and antagonists. Additionally, the involvement of increased calcium influx should also be analyzed.

Response 1:

Thank you for this precious and valuable comment. We have conducted a series of additional experiments to establish the mechanism that upregulation of PIEZO1 induced by simulated microgravity increased CXCR4 protein level through Ca²⁺ influx. Firstly, we have proved that the expression levels of CXCR4 protein increased significantly under simulated microgravity, but decreased after knocking down PIEZO1.Please see Figure 4A and Page 7, lines 182-185 in the revised manuscript. Secondly, we used Fluo-4AM staining to detect the intracellular Ca²⁺ of HUVECs. Exposure to simulated microgravity caused a significant increase in Ca²⁺ levels of HUVECs, which was dramatically attenuated by PIEZO1 knockdown treatment. Please see Figure 4B and Page 7, lines 187-192 in the revised manuscript. Then we used a permeable calcium chelator (BAPTA‐AM) to investigate the effects of Ca²⁺ levels on expressions of CXCR4 in HUVECs under simulated microgravity. Western Blot analysis showed BAPTA‐AM treatment significantly reduced the elevation of CXCR4 protein levels induced by simulated microgravity. Please see Figure 4C, Page 7, lines 192-196 in the revised manuscript. Taken together, these findings suggest that PIEZO1 significantly enhanced the expression of CXCR4 via Ca²⁺ influx under simulated microgravity.

As for PIEZO1 antagonists, we used GsMTx4(2.5 µM) to treat HUVECs under simulated microgravity. Consistent with the results of PIEZO1 knockdown, GsMTx4 treatment reduced the elevated CXCR4 expression induced by simulated microgravity, as shown in the figure. This further substantiates the notion that PIEZO1 enhanced the expression of CXCR4 in endothelial cells under simulated microgravity.

Comments 2: It is preferable to discuss the mechanism by which microgravity increased PIEZO1 expression. Additionally, while FGF-2 and VEGF are well-known growth factors responsible for vascular endothelial proliferation and migration, it would be beneficial to also consider the significance of chemokine receptor involvement.

Response 2:

We greatly appreciate your insightful suggestions and constructive feedback on our manuscript. We have expanded our discussion to include a more in-depth analysis of the possible mechanisms through which microgravity may increase PIEZO1 expression. Please see Page 11, lines 239-246 in the revised manuscript.

Furthermore, we have tested FGF-2 and VEGF expression level. Western blot analysis showed that the expression of FGF-2 and VEGF in endothelial cells did not change following simulated microgravity, see following figure. This observation suggests that VEGFA and FGF-2 may not be involved in the migration of endothelial cells induced by simulated microgravity. Endothelial cell migration is a complex process involving the coordination of multiple molecules. Our previous research has shown that mitophagy inhibits endothelial migration by reducing the high expression of MMP1 under simulated microgravity conditions[1]. In this study, we have expanded upon the role of the PIEZO1/Ca2+/CXCR4 signaling axis in mediating the effects of simulated microgravity on endothelial cell migration. Our study is the first to demonstrate that simulated microgravity promotes endothelial cell migration through the activation of this signaling pathway. And This finding underscores the intricate regulatory mechanisms at play during endothelial cell migration.

Comments 3: Regarding Figure 1B, the expression of GAPDH protein was significantly changed by microgravity in a time-dependent manner, making it difficult to determine if the Western blot analysis was performed correctly. The data should either be replaced or another internal control should be used. Additionally, it appears there are two bands for PIEZO1; it should be clarified which one corresponds to PIEZO1.

Response 3:

    Thank you for your meticulous review and for pointing out the concerns regarding Figure 1B in our manuscript. GAPDH is widely used as an internal control in studies involving simulated microgravity[1,2], and its expression doesn’t change under these conditions. The variation in GAPDH expression observed in Figure 1B is likely due to variations in sample loading and experimental methods that led to inconsistent normalization. We acknowledge that this could potentially mislead the interpretation of the data.

Regarding the two bands observed for PIEZO1, we suspect that the additional band may be due to non-specific binding of the antibody. To avoid any ambiguity and to ensure the highest standards of data quality, we have conducted a thorough re-evaluation of the Western blot analysis, ensuring proper sample loading and standardized procedures to maintain consistency in the internal control. The results from these experiments have been used to replace Figure 1B in the revised manuscript.   

Comments 4: Regarding Figure 4A and 4C, it should be specified which protein band is being referred to.

Response 4:

Thank you for your meticulous review and for pointing out the oversight regarding the labeling of the bands in our Western blot images. We sincerely apologize for this lapse and appreciate the opportunity to correct it. The bands in question have now been clearly labeled in the revised manuscript. We have also taken this opportunity to thoroughly check all other figures and data for accuracy and completeness.

Comments 5: Regarding Figure S1, the Figure S1 and the legend do not correspond correctly and should be revised.

Response 5:

I would like to express my sincere gratitude for your insightful review and for bringing to our attention the issues with the figure captions. We apologize for any confusion that our initial submission may have caused. Following your valuable suggestions, we have made the necessary corrections to the problematic figure captions in the revised supplementary (Please see Figure S1). We have ensured that all figure captions are now clear, accurate, and properly reflect the content of the images they accompany.

Comments 6: Line 16: PIZEO1 → PIEZO1

Response 6:

I am writing to convey my deepest apologies once again for the oversight in our previous submission. We appreciate your patience and guidance. As per your instructions, we have made the necessary revisions to our manuscript.

    We are committed to upholding the integrity and clarity of our research, and your feedback has been instrumental in helping us achieve that. We hope that these revisions will meet with your approval and that our manuscript will be considered for publication.

Once again, thank you for your constructive criticism and for the opportunity to to address the issues you have identified.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding highlighted changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: It almost looks like PIEZO1 exists as a doublet as evidenced in the difference in band size in figure 1 B. Was this taken into account/verified in any form of analyses?

Response 1:

Thank you for your careful review and for pointing out the potential issue with the band pattern observed in Figure 1B of our manuscript.

We would like to confirm that PIEZO1 is not a doublet; rather, it has a homotrimeric structure, resembling a trident or three-leaf propeller. This trimeric assembly is fundamental to its function as a mechanosensitive ion channel[1].To address this, we have repeated the Western blot experiments with greater stringency in sample preparation and antibody selection. The new data have resolved the issue and confirm the expression of PIEZO1 as a single band corresponding to its expected molecular weight. We have replaced Figure 1B in the revised manuscript with the new, more accurate figure. Please see Figure 1B in the revised manuscript. Please see Figure 1 B and Page 3 in the revised manuscript.

Comments 2: The authors utilize a number of different time points throughout the study as exemplars of certain conditions within the 6–48-hour range. Is there a reason why certain timepoints were selected in certain instances? Were the assays such as migration and wound healing performed at other timepoints or only those presented?

Response 2:

We greatly appreciate your thorough review and valuable comments on our manuscript. Our study employed a 2D clinostat to simulate the short-term microgravity effects on cells. Due to the limited oxygen in the rotating chamber, the maximum duration for continuous rotation is 72 h, and in general, 48h is used for rotation. While our previous studies have utilized a 48-hour simulated microgravity period[2,3], the current study selected 6-hour, 12-hour, 24-hour, and 48-hour time points to further elucidate the expression patterns of PIEZO1 following simulated microgravity conditions.

For the Transwell and wound healing assays, we conducted our assessments at the 12-hour, without performing these assays at other time points. This decision was primarily based on the following considerations:

1.Literature Reference: Our approach aligns with relevant literature, which has adopted the 12-hour time point for such assessments, providing us with a reliable experimental framework[4,5].

2.Experimental Conditions: During the experimental process, we used a low-serum medium to mitigate the impact of cell proliferation on the results. In contrast, by the 24-hour mark, we observed a considerable degree of cell detachment, which could potentially skew the results of the assays. The integrity and viability of the cells at the 12-hour time point were significantly better, making it the most appropriate choice for conducting our assays.

Comments 3: The western blot in Figure 4 A and C need labelling.

Response 3:  

Thank you for your meticulous review and for pointing out the oversight in our manuscript. We apologize for the missing labels in the Western blot images of Figure 4 A and C. The bands in question have now been clearly labeled in the re-submitted manuscript. We have also taken this opportunity to thoroughly check all other figures and data for accuracy and completeness.

Comments 4：In reviewing the original blot files some of the antibodies appear to detect several non-specific bands. In particular, CXCR4. Did the authors perform any kind of QC to ensure the band they selected for analysis was in fact that which they expected?

Response 4: 

Thank you for your careful review and insightful comments regarding our manuscript. We have taken note of the issue you raised concerning the non-specific bands detected by some antibodies, particularly CXCR4, in the original blot files. We acknowledge your concern and have conducted a thorough analysis to address this matter. Upon further examination, we believe that the observed non-specific binding could be attributed to the inherent properties of the antibodies used. However, we are confident that the band we selected for analysis in our manuscript is indeed the target band.

To substantiate our claim, we referred to the antibody's product datasheet. According to the specifications provided, the CXCR4 band is expected to be around 43 kDa. Upon reviewing the original blot images, we observed that the predominant band aligns with this size, which is consistent with the expected molecular weight as per the datasheet.

Comments 5： The signal presented in Figure 4B appears much stronger than what is represented in the bar chart. Can the authors clarify how they measured this signal and processed the results?

Response 5:     

Thank you for your attention to the details in our manuscript, particularly regarding the discrepancy between the signal intensity in Figure 4B and the bar chart representation. We appreciate the opportunity to clarify our methods and results.

In our study, the experiments were conducted in three groups: Con+si-NC, MG+si-NC, and MG+si-PIEZO1. Each group consisted of three independent samples, and for each sample, we captured three different fields of view. To measure the signal, we utilized the Image J software to analyze the average fluorescence intensity of each group's samples. Following the quantification, we employed GraphPad Prism software to perform the statistical analysis on the collected data. The bar chart in the manuscript presents the final statistical results, which are the average fluorescence intensities of each group. The images shown in Figure 4B was selected to illustrate the most significant changes observed in one of the fields of view, which may account for the stronger signal appearance compared to the average values depicted in the bar chart. To avoid any ambiguity, we have replaced the figure with a more representative one. Please see Figure 4 B and Page 8 in the re-submitted manuscript

We hope this explanation addresses your concerns and provides a clear understanding of our measurement and processing methods.

Comments 6： The scratches etched into the monolayers appear to be different sizes based on the lines drawn – did the authors take this into account in their analyses? How were the calculations performed to normalize these rates of closures to one another?

Response 6:

Thank you for your close examination of our manuscript and for your specific inquiry about the scratch assay results presented in our study.

We acknowledge that there may have been some inconsistencies in the scratch width due to potential operational errors during the process. The scratches were made using a 200 µl pipette tip, and variations could have occurred.

To address this, we have calculated the scratch healing rate using the formula: scratch width at 0 hour - scratch width at 12 hours. This method allowed us to normalize the rates of closure despite the initial differences in scratch sizes. We believe this approach provides a fair assessment of the healing process over the 12-hour period.

Furthermore, to ensure clarity and to avoid any ambiguity in our presentation, we have replaced the original images with ones that are more consistent and representative of our experimental findings. We have also included a detailed description of our scratch assay methodology and the normalization process in the revised manuscript.

Comments 7: How was the working concentration of the CXCR4 inhibitor optimized? Is there an assay the authors performed to confirm that this compound was having the desired effect at the concentration used?

Response 7:

Thank you for your inquiry regarding the optimization of the working concentration of the CXCR4 inhibitor and the validation of its effect at the used concentration. AMD3100 is a selective CXCR4 antagonist that does not affect the expression levels of the CXCR4 receptor. Currently, there are no reports in the literature regarding the effective concentration of CXCR4 antagonists. Without objective indicators, the effectiveness of the antagonist can only be detected by observing downstream biological processes.

In our study, we conducted a thorough literature review and found a broad range of concentrations reported for AMD3100, spanning from 5 µM to 700 µM[6^,7]. To determine the appropriate concentration for our experiments, we selected a middle-ground concentration within this range. We chose 50 µM, which is a concentration that has been widely used in the literature for its effectiveness and lack of overt negative effects on cell morphology and growth[8^,9].

At the 50 µM concentration, we observed a significant inhibition of endothelial cell migration under simulated microgravity conditions, without any apparent adverse effects on cell morphology or proliferation. This concentration has been employed in various studies, demonstrating its reliability and consistency in modulating CXCR4 activity.

We believe that our selection of the 50 µM concentration for AMD3100 is well-justified and supported by both the literature and our experimental findings. We hope this response adequately addresses your concerns and provides a clear understanding of our approach to optimizing the working concentration of the CXCR4 inhibitor.

Comments 8:  When the microgravity environment was being simulated was the culture vessel completed filled? How were the coverslips kept in place to ensure they weren’t subjected to movement as the clinostat operated?

Response 8: 

Thank you for your inquiry about the simulation of the microgravity environment in our study. To simulate the effects of microgravity, we employed a two-dimensional (2D) clinostat, which was specifically developed and provided by the China Astronaut Research and Training Center (A). Human Umbilical Vein Endothelial Cells were incubated on 2.55 × 2.15 cm coverslips within the chamber filled with culture medium. Each chamber contains four coverslips, which are carefully inserted into specially designed rackets to ensure that they remain stable and do not move during the rotation process(B). In order to avoid the influence of shear stress, all culture flasks were meticulously filled with medium to eliminate the presence of air bubbles and were hermetically sealed during rotation(C).

The clinostat operates by rotating the cells around a horizontal axis at a speed of 30 rpm, which effectively randomizes the gravitational vector (D). This method is designed to mimic the microgravity conditions of low Earth orbit, which is approximately equivalent to 10^-3 g of gravitational force. For the control group, the cells were cultured under the same conditions as the experimental group, with the exception of clinorotation, ensuring that any observed effects could be attributed to the simulated microgravity environment.

Comments 9: What was the source of the primer sequences, and did the authors verify the primers were MIQE guidelines compliant?

Response 9:  

Thank you for your inquiry regarding the source of our primer sequences and their compliance with the MIQE guidelines. The primer sequences utilized in our study were sourced from the PrimerBank website, a reputable database that provides reliable and validated primers for various genes. For the genes of interest in our study, PIEZO1 and GAPDH, we ensured that the primers adhered to the following criteria in accordance with the MIQE guidelines: The GC content of both the PIEZO1 and GAPDH primers was maintained within the 40-60% range, which is essential for achieving optimal amplification efficiency and specificity. The melting temperatures (Tm) of the primers were confirmed to fall within the 50-65°C interval, ensuring that the primers would anneal effectively under standard PCR conditions. The PIEZO1 primers were designed to span exons 38 and 39, while the GAPDH primers spanned exons 8 and 9. This exon-spanning design helps to prevent the amplification of potential genomic DNA contamination. To further verify the specificity of our primers, we conducted sequence alignment using the NM sequences from the NCBI database. The results of this alignment confirmed that our primers were specific to their target genes, with no significant homology to other genomic regions.

In summary, our primer design and selection process were meticulous and strictly followed the principles outlined in the MIQE guidelines[10]. We are confident that the primers used in our study are both specific and reliable, contributing to the validity and reproducibility of our research findings.

Comments 10: More details on the image analyses of the microscopy images are warranted. Did the authors image several fields? Were they selected at random by the software? Etc.

Response 10:

Thank you for your request for additional details regarding the image analysis of our microscopy images. We conducted three independent repetitions for each of the three experimental groups, with three samples per group. Each sample was randomly captured in at least three different fields of view for subsequent statistical analysis. In all cases, the image acquisition was performed in a blinded manner to minimize bias. The selection of fields was automated by the software to ensure randomness, and the analyses were conducted without prior knowledge of the experimental conditions.

We hope this information provides the necessary details for your assessment and addresses your concerns about the image analysis process.

Comments 11: Did the authors examine the effect of a nonspecific siRNA on PIEZO1 expression levels?

Response 11:   

Thank you for your inquiry about the examination of the effect of a nonspecific siRNA on PIEZO1 expression levels.

In the supplementary (Figure S1),we have included additional data that demonstrate our evaluation of three different siRNAs targeting PIEZO1. Through this assessment, we observed the extent of knockdown at both the mRNA and protein levels for each sinuate careful analysis, we selected the siRNA3 that exhibited the most pronounced reduction in PIEZO1 expression for our subsequent experiments. We believe that the inclusion of these data in the supplementary addresses the issue of siRNA specificity and strengthens the validity of our findings. We hope this information satisfies your query and provides the necessary evidence of the specificity of our siRNA approach.

Comments 12:  As the wound healing measurements were performed in normal gravity as is interpreted from the methods section, how can the authors be sure the effects of microgravity were sustained and reflected in the microgravity conditioned cells in the 12 hours after the assay began?

Response 12:

We appreciate your concern about ensuring that the effects of microgravity are sustained and reflected in the microgravity-conditioned cells during the 12 hours following the initiation of the assay. We maintained strict experimental conditions for both the simulated microgravity group and the control group, with the only variable being the rotation to simulate microgravity. Our experimental design ensured that all other conditions, including the culture medium, cell seeding density, and incubation conditions, were identical for both groups. This rigorous control of variables allows us to confidently attribute any observed increases in endothelial cell migration to the simulated microgravity conditions. Furthermore, our assertion that the enhanced migration is due to simulated microgravity is supported by previous research conducted in our laboratory. Our previous studies have demonstrated similar effects of simulated microgravity on endothelial cells, which corroborates the findings observed in the current study[3,11].

We believe that the consistency in our experimental approach, combined with the supportive evidence from our previous research, provides a solid foundation for concluding that the effects observed in our study are indeed a result of the simulated microgravity.

We hope this explanation clarifies how we ensured the sustained effects of microgravity in our study and addresses your concerns.

Thank you once again for your insightful comments and for giving us the opportunity to improve our work.

Response to Reviewer 3Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding highlighted changes in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In reviewing the manuscript and the included blots for each still draw questions. For example, the blots performed for each examination of PIEZO1 are inconsistent with one another. The authors mention repeating the Western blot to address the original points raised, however, in reviewing those submitted with the article the bands obtained look varied between all included. For example, in some blots the band is almost like a smear and/or multiple bands are present, whereas in other the blots are much cleaner. I appreciate the authors are referring to the data sheet, but often these antibody checks have been performed using a particular cell line, and in such instances, every antibody should be validated for each respective model/system being employed regardless of the data sheet info. Have the authors ever included a positive/negative control to validate the bands they are analysing?

Response 1:

Thank you for your thorough review and constructive comments on our manuscript. We appreciate the concerns raised regarding the consistency of the Western blot results for PIEZO1. We assure you that all our results are reproducible and we have taken your feedback seriously to address the issues highlighted.

We acknowledge that the variability observed in the PIEZO1 blots may be attributed to methodological variations during sample collection. To rectify this, we have recollected the samples and ensured a standardized protocol. Given that PIEZO1 is a membrane protein, we have adopted a milder denaturation approach to preserve its integrity during Western blot analysis. Regarding the presence of non-specific bands, we have included si-PIEZO1 samples as negative controls to validate the bands of interest. Upon re-examination, we found that these non-specific bands remained unaffected by the knockdown, suggesting that they are likely due to non-specific antibody interactions rather than representing PIEZO1 protein. Please see Figure S1 in the supplementary.

We understand the importance of validating each antibody for the respective model or system used, regardless of the information provided on the data sheet. We have taken additional steps to ensure that all antibodies are rigorously tested and validated for the cell lines and models employed in our study.

We hope that these revisions and additional controls will address the concerns raised and strengthen the validity of our findings. We are committed to maintaining the highest standards of scientific integrity and thank you once again for your insightful comments.

Comments 2: For MIQE guidelines validation, there are other aspects of primers that need validation. For example, clean single peak melt curves, product size validation, and efficiency falling between 90%-105% to name a few. Were any of these checks performed?

Response 2:

Thank you for your meticulous review and for bringing to our attention the MIQE guidelines for primer validation. We appreciate the opportunity to clarify the measures we have taken to ensure the quality of our primers in our study. Before conducting our experiments, we meticulously ensured the quality of our primers, which is reflected in several key aspects:

Firstly, we have examined the melt curves for all our primers and showed that they all exhibit clean, single peaks, indicating the specificity of our primers and the absence of primer-dimers in our reactions(A).

Secondly, to validate the product size, we have utilized agarose gel electrophoresis to analyze the amplicons. The results confirm that the GAPDH product is 131 base pairs (bp) and the PIEZO1 product is 127 bp, which are in accordance with the predicted sizes (BC).

Furthermore, we have calculated the amplification efficiency for both primers. The GAPDH primers show an efficiency of 91.5%, and the PIEZO1 primers an efficiency of 103%. Both values fall well within the recommended range of 90% to 110%(D).

In summary, these validations confirm that our primers are suitable for use in our experiments, ensuring the accuracy and reproducibility of our qPCR data.

We appreciate your guidance and are grateful for the opportunity to enhance the rigor of our study.

We hope that these revisions and additional controls will address the concerns raised and strengthen the validity of our findings. We are committed to maintaining the highest standards of scientific integrity and thank you once again for your insightful comments.