Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome

Endothelial dysfunction caused by endothelial cell injuries is the initiating factor for atherosclerosis (AS), and lipid peroxidative injury is one of a dominant factor for AS pathogenesis. Using RNA-seq, we compared changes in transcriptome expression before and after endothelial cell injury, and found 311 differentially expressed genes (DEGs), of which 258 genes were upregulated and 53 genes were downregulated. The protein–protein interactions (PPIs) between the genes were analysed using the STRING database, and a PPI network of DEGs was constructed. The relationship distributions among these PPIs were analysed by performing network node statistics. We found that in the top 20 DEGs with high connected protein nodes in the PPI network, 16 were upregulated and 4 were downregulated. Gene ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis on the DEGs were also performed. By comparing the upregulated expressed genes with high connected protein nodes in the PPI network to those related to endothelial cell lipid damage and repair in the GO analysis, we identified seven genes (NOX4, PPARA, CCL2, PDGFB, IL8, VWF, CD36) and verified their expression levels by real-time polymerase chain reaction. The protein interactions between the seven genes were then analysed using the STRING database. The results predicted that CCL2 interacts with NOX4, PPARα, PDGFβ and VWF individually. Thus, we examined the protein expression levels of CCL2, NOX4, PPARα, PDGFβ and VWF, and found that the expression levels of all proteins were significantly upregulated after the lipid peroxidative injury, with CCL2 and PPARα exhibiting the highest expression levels. Therefore, we investigated the interregulatory relationship between CCL2 and PPARα and their roles in the repair of endothelial cell injury. With the help of gene overexpression and knockdown techniques, we discovered that PPARα promotes the repair of endothelial cell injury by upregulating CCL2 expression in human umbilical vein endothelial cells but that CCL2 cannot regulate PPARα expression. Therefore, we believe that PPARα participates in the repair of endothelial cell lipid peroxidative injury through regulating the expression of CCL2.

PPARα activated downstream CCL2 expression in HUVEC cells and in ApoE-/-mice model. By comparison of proliferation, migration and angiogenesis in HUVEC cells with modified PPARα activity and CCL2 expression, they found that the function of PPARα during the repair of endothelial cell injury is dependent on CCL2. Thus, this manuscript uncovered the role of PPARα-CCL2 during the repair of lipid injury. However, the authors could strengthen their finding in ApoE-/-mice model. It seems that the "damage stage" and "repair stage" after endothelial cell injury was confused (see below). In addition, the experimental rationales should be improved. These points prevent me from recommending the manuscript for publication unless the following concerns could be carefully addressed.
Major points: 1. Page 11, for readers' convenience, the authors should briefly introduce the aim of the study and the workflow (cell/animal model, sample collection and data analysis) at the beginning of the "Results" section, although part of these information has been provided in "Materials and Methods". 2. Figure 1, the authors used HUVEC cells treated with 200 μg/mL of oxLDL for 24 hours as cellular model of endothelial injury. The time point after injury is important for cellular response (acute, chronic response/repair stages) to injury/stress. Have the authors optimized with different time points and oxLDL doses, and verified the in intro vascular endothelial cell injury model? 3. Figure 1, the authors have successfully established animal model (ApoE-/-mice fed with a high-fat diet), they could cross validate their RNA-seq results from cell model vs animal model. 4. Figure 2, the rationale is not clear from RNA-Seq to PPIs network, and back to validation of mRNA expression. The authors should also consider subcellular localization to improve the reliability of the predicted PPIs, e.g. Figure 2 predicted that CCL2 (extracellular or membrane) and PPARα (nuclear) are interacted, they are assumed to be co-localized. 5. Figure 3, the rationale is not clear to choose CCL2 and PPARα to "speculate CCL2 and PPARα play critical roles in the injury repair caused by lipid peroxidation" and ignore PDGFB, NOX4, VWF while all of them shared similar expression pattern after lipid injury. 6. Figure 4B, RT-qPCR of CCL2 is suggested to distinguish whether CCL2 is regulated by PPARα at transcription or post-transcription level. Prediction of PPARα consensus binding site in the promoter region of CCL2 gene is suggested to distinguish whether CCL2 is regulated by PPARα directly or indirectly. 7. Figure 4C and page 13, the authors should briefly introduce animal models used here (ApoE-/normal diet mice as control group and ApoE-/-high-fat diet mice as model group), although mentioned in "Materials and Methods". Have the authors extracted ventricular outflow tract tissues to quantify CCL2 expression among control, model, PPARα agonist and PPARα antagonist groups? 8. Page 13 Line 4, the authors mentioned that "we found a small amount of CCL2 expression in the control group and the model group", this indicated that there are no up-regulation of CCL2 in mice model of lipid injury, while data from cell model (oxLDL for 24 hrs, without PPARα agonist) showed induction of CCL2 and PPARα. Could the author explain the discrepancy is due to different model or different stages (damage, repair) after lipid injury? The authors should also investigate PPARα expression among these animal models because the activation of PPARα maybe a hallmark of repair stage. 9. Figure 6, the CCL2 expression level should be included in parallel with the effect of PPARα on proliferation, migration and angiogenesis. The control group (without oxLDL) should also be included.
Minor points: 1. Pages 9 line 16, "Sections were washed, mounted, and examined using a light microscope", please check whether "light microscope" is correct for examination of immunofluorescence staining.
2. Figure 4C, the labeling of "control" and "model" can not distinguish animal model used here from cell model used elsewhere. The authors could label with "ApoE-/-mice control" and "ApoE-/-mice model". 3. Table 1 is not necessary. The authors should provide the details of RNA-seq results (related to Figure 1) including names of total 311 genes and fold changes into Supplemental Table 1.

19-Aug-2019
Dear Professor Chen, We are writing to inform you that the Editor has reached a decision on your manuscript RSOB-19-0141 entitled "Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome", submitted to Open Biology.
As you will see from the reviewer's comments below, there are a number of criticisms that prevent us from accepting your manuscript at this stage. The reviewer suggests, however, that a revised version could be acceptable, if you are able to address their concerns. If you think that you can deal satisfactorily with the reviewer's suggestions, we would be pleased to consider a revised manuscript.
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Sincerely, The Open Biology Team mailto: openbiology@royalsociety.org Reviewer(s)' Comments to Author(s): Referee: 1 Comments to the Author(s) In the current manuscript by Dou et al. entitled "Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome", the authors identified differentially expression genes after endothelial cell injury in HUVEC cells via RNA-seq. The predicted PPIs network suggested that CCL2 may play a central role after endothelial cell injury. Investigation into CCL2 and PPARα revealed that PPARα activated downstream CCL2 expression in HUVEC cells and in ApoE-/-mice model. By comparison of proliferation, migration and angiogenesis in HUVEC cells with modified PPARα activity and CCL2 expression, they found that the function of PPARα during the repair of endothelial cell injury is dependent on CCL2. Thus, this manuscript uncovered the role of PPARα-CCL2 during the repair of lipid injury. However, the authors could strengthen their finding in ApoE-/-mice model. It seems that the "damage stage" and "repair stage" after endothelial cell injury was confused (see below). In addition, the experimental rationales should be improved. These points prevent me from recommending the manuscript for publication unless the following concerns could be carefully addressed.
Major points: 1. Page 11, for readers' convenience, the authors should briefly introduce the aim of the study and the workflow (cell/animal model, sample collection and data analysis) at the beginning of the "Results" section, although part of these information has been provided in "Materials and Methods". 2. Figure 1, the authors used HUVEC cells treated with 200 μg/mL of oxLDL for 24 hours as cellular model of endothelial injury. The time point after injury is important for cellular response (acute, chronic response/repair stages) to injury/stress. Have the authors optimized with different time points and oxLDL doses, and verified the in intro vascular endothelial cell injury model? 3. Figure 1, the authors have successfully established animal model (ApoE-/-mice fed with a high-fat diet), they could cross validate their RNA-seq results from cell model vs animal model. 4. Figure 2, the rationale is not clear from RNA-Seq to PPIs network, and back to validation of mRNA expression. The authors should also consider subcellular localization to improve the reliability of the predicted PPIs, e.g. Figure 2 predicted that CCL2 (extracellular or membrane) and PPARα (nuclear) are interacted, they are assumed to be co-localized. 5. Figure 3, the rationale is not clear to choose CCL2 and PPARα to "speculate CCL2 and PPARα play critical roles in the injury repair caused by lipid peroxidation" and ignore PDGFB, NOX4, VWF while all of them shared similar expression pattern after lipid injury. 6. Figure 4B, RT-qPCR of CCL2 is suggested to distinguish whether CCL2 is regulated by PPARα at transcription or post-transcription level. Prediction of PPARα consensus binding site in the promoter region of CCL2 gene is suggested to distinguish whether CCL2 is regulated by PPARα directly or indirectly. 7. Figure 4C and page 13, the authors should briefly introduce animal models used here (ApoE-/normal diet mice as control group and ApoE-/-high-fat diet mice as model group), although mentioned in "Materials and Methods". Have the authors extracted ventricular outflow tract tissues to quantify CCL2 expression among control, model, PPARα agonist and PPARα antagonist groups? 8. Page 13 Line 4, the authors mentioned that "we found a small amount of CCL2 expression in the control group and the model group", this indicated that there are no up-regulation of CCL2 in mice model of lipid injury, while data from cell model (oxLDL for 24 hrs, without PPARα agonist) showed induction of CCL2 and PPARα. Could the author explain the discrepancy is due to different model or different stages (damage, repair) after lipid injury? The authors should also investigate PPARα expression among these animal models because the activation of PPARα maybe a hallmark of repair stage. 9. Figure 6, the CCL2 expression level should be included in parallel with the effect of PPARα on proliferation, migration and angiogenesis. The control group (without oxLDL) should also be included.
Minor points: 1. Pages 9 line 16, "Sections were washed, mounted, and examined using a light microscope", please check whether "light microscope" is correct for examination of immunofluorescence staining. 2. Figure 4C, the labeling of "control" and "model" can not distinguish animal model used here from cell model used elsewhere. The authors could label with "ApoE-/-mice control" and "ApoE-/-mice model". 3. Table 1 is not necessary. The authors should provide the details of RNA-seq results (related to Figure 1) including names of total 311 genes and fold changes into Supplemental

Comments to the Author
The authors have substantially improved the revised version of this manuscript and have sufficiently addressed my previous concerns. I have now only a few minor concerns: 1. Page 13, lines 1-18 (the paragraph before section 3.1) seem a summary for this study and are unusual, weird in regards to paper format. In fact, a brief introduction of the background of transcriptome analysis in this study is sufficient for understanding by the readers. I would suggest moving lines 1-7 to the beginning of section 3.1, while lines 8-18 could either move to the end of section 1 (Introduction) or be deleted. 2. To avoid of confusion, I would suggest adding the following (or something like that) at the end of section 3.1: The interactions between PPI-predicted proteins include direct (physical) and indirect (functional) associations. 3. Sections 3.2 and 3.3 could be combined. Section 3.5 could be split into 2 sections: one is related to Fig. 4 (PPARα regulates CCL2 expression) and another is related to Fig. 5 and 6 (PPARα participates in the repair of cell injury induced by lipid peroxidation through regulating CCL2 expression).

11-Nov-2019
Dear Professor Chen We are pleased to inform you that your manuscript RSOB-19-0141.R1 entitled "Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome" has been accepted by the Editor for publication in Open Biology. The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the reviewer(s)' comments and revise your manuscript.
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Data accessibility section To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: 1. Page 13, lines 1-18 (the paragraph before section 3.1) seem a summary for this study and are unusual, weird in regards to paper format. In fact, a brief introduction of the background of transcriptome analysis in this study is sufficient for understanding by the readers. I would suggest moving lines 1-7 to the beginning of section 3.1, while lines 8-18 could either move to the end of section 1 (Introduction) or be deleted. 2. To avoid of confusion, I would suggest adding the following (or something like that) at the end of section 3.1: The interactions between PPI-predicted proteins include direct (physical) and indirect (functional) associations. 3. Sections 3.2 and 3.3 could be combined. Section 3.5 could be split into 2 sections: one is related to Fig. 4 (PPARa regulates CCL2 expression) and another is related to Fig. 5 and 6 (PPARa participates in the repair of cell injury induced by lipid peroxidation through regulating CCL2 expression).

15-Nov-2019
Dear Professor Chen We are pleased to inform you that your manuscript entitled "Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome" has been accepted by the Editor for publication in Open Biology.
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Thank you for your fine contribution. On behalf of the Editors of Open Biology, we look forward to your continued contributions to the journal. Investigation into CCL2 and PPARα revealed that PPARα activated downstream CCL2 expression in HUVEC cells and in ApoE-/-mice model. By comparison of proliferation, migration and angiogenesis in HUVEC cells with modified PPARα activity and CCL2 expression, they found that the function of PPARα during the repair of endothelial cell injury is dependent on CCL2. Thus, this manuscript uncovered the role of PPARα-CCL2 during the repair of lipid injury. However, the authors could strengthen their finding in ApoE-/-mice model. It seems that the "damage stage" and "repair stage" after endothelial cell injury was confused (see below). In addition, the experimental rationales should be improved. These points prevent me from recommending the manuscript for publication unless the following concerns could be carefully addressed.

ANSWER:
Dear editor and reviewer, thank you very much for giving us the opportunity to revise the manuscript, we will provide point-by-piont response to the comments and resubmit the improved manuscript online.
Major points: 1. Page 11, for readers' convenience, the authors should briefly introduce the aim of the study and the workflow (cell/animal model, sample collection and data analysis) at the beginning of the "Results" section, although part of these information has been provided in "Materials and Methods".
Answer 1: Dear reviewer, thanks for your advice firstly, we have added brief Appendix A introduction of the aim of the study and the workflow at the beginning of the "Results" section in revised manuscript, please check. Figure 1,  of cell showed positive oil red O staining at 24 hr only, we did not detect the difference between injury group and control group at 6 hr and 12 hr, at 48 hr, there was no significant difference in cell viability, proliferation and apoptosis between the ox-LDL group and the control group. 300 μg/mL ox-LDL resulted in more than 50% cell apoptosis. Therefore, we chosen 200 μg/mL as our final concentration. Figure 1, the authors have successfully established animal model (ApoE-/-mice fed with a high-fat diet), they could cross validate their RNA-seq results from cell model vs animal model.

3.
Answer 3: Dear reviewer, thank you very much for your advice, since the cells we are using is human umbilical vein endothelial cells, although transcripts of human and mouse have high homology, different transcripts may transcribe proteins with different functional sites. Therefore, we did not cross validate their RNA-seq results from cell model vs animal model. However, we believe you gave us a good suggestion, we will use a mouse vascular endothelial cell to make a damage model, and compare the RNA-seq results with the results of Apoe -/mice to obtain a more reliable conclusion. Figure 2, the rationale is not clear from RNA-Seq to PPIs network, and back to validation of mRNA expression. The authors should also consider subcellular localization to improve the reliability of the predicted PPIs, e.g. Figure 2 predicted that CCL2 (extracellular or membrane) and PPARα (nuclear) are interacted, they are assumed to be co-localized.

4.
Answer 4: Dear reviewer, thanks for your professional advice firstly. As you said, there is no direct connection between RNA-seq and PPI network. RNA-seq is only used to detect the expression and changes of a molecular at the RNA level, changes of protein level are not consistent with changes in RNA expression, and PPI network prediction is only a speculation of the relationship between proteins according to the results of RNA-seq. The interactions between PPI-predicted proteins include direct physical interactions and indirect correlations. CCL2 is a chemokine, and chemokines mostly perform its function as secreted proteins, PPARα is a transcription factor, it exerts regulatory functions by binding to the promoter region of the target protein, when the PPI network predicts that there is an interaction between CCL2 and PPARα, we speculate that PPARα binds to the promoter region of CCL2 and regulates the expression of CCL2. Therefore, the PPI network is just a tool we use to speculate whether there is interaction between proteins, and we need verified experiments to proving the authenticity of this speculation. Figure 3, the rationale is not clear to choose CCL2 and PPARα to "speculate CCL2 and PPARα play critical roles in the injury repair caused by lipid peroxidation" and ignore PDGFB, NOX4, VWF while all of them shared similar expression pattern after lipid injury.

5.
Answer 5: Dear reviewer, thank you very much for your valuable comments on the issues in our manuscript. Because the interactions between PPI-predicted proteins include direct physical interactions and indirect correlations, by analyzing each protein-related function, PDGFβ and VWF can be used as marker proteins of endothelial cells, and as the main protease producing reactive oxygen species, NOX4 is usually in a highly active state after endothelial cell injury, their interaction with the chemokine CCL2 may not be a direct regulatory relationship, and PPARA, as a transcription factor, might directly bind to the promoter region of CCL2 to regulate its expression. Therefore, we firstly selected PPARA and CCL2 as research targets.
Based on your suggestions, we will continue to study the relationship between NOX4, PDGFβ, VWF and CCL2, respectively. Figure 4B, RT-qPCR of CCL2 is suggested to distinguish whether CCL2 is regulated by PPARα at transcription or post-transcription level. Prediction of PPARα consensus binding site in the promoter region of CCL2 gene is suggested to distinguish whether CCL2 is regulated by PPARα directly or indirectly.

6.
Answer 6: Dear reviewer, thank you very much for your professional advice.
RT-qPCR assay of CCL2 has been performed and proved that CCL2 was regulated by PPARα at transcription level, and luciferase assay confirmed that PPARα could bind to the promoter region of CCL2 gene to promote CCL2 expression. These results and experiment mothods (2.6 Luciferase reporter assay) have been added to figure 4 and to manuscript, please check. Figure 4C and page 13, the authors should briefly introduce animal models used here (ApoE-/-normal diet mice as control group and ApoE-/-high-fat diet mice as model group), although mentioned in "Materials and Methods". Have the authors extracted ventricular outflow tract tissues to quantify CCL2 expression among control, model, PPARα agonist and PPARα antagonist groups?

7.
Answer 7: Dear reviewer, thanks for your professional advice. We have gave a brief introduction of animal model in page 13 and figure 4, and we also examined the expression of CCL2 of the left ventricular outflow tract tissues in Apoe -/mice, please check.

8.
Page 13 Line 4, the authors mentioned that "we found a small amount of CCL2 expression in the control group and the model group", this indicated that there are no up-regulation of CCL2 in mice model of lipid injury, while data from cell model (oxLDL for 24 hrs, without PPARα agonist) showed induction of CCL2 and PPARα.
Could the author explain the discrepancy is due to different model or different stages (damage, repair) after lipid injury? The authors should also investigate PPARα expression among these animal models because the activation of PPARα maybe a hallmark of repair stage.
Answer 8: Dear reviewer, thank you for pointing out the flaws in the manuscript. The description of this sentence "we found a small amount of CCL2 expression in the control group and the model group" may lead to some misunderstanding, the intention of this sentence is to show that the expression of CCL2 in the Apoe -/mice with normal diet and high fat diet is relatively small compared with the Apoe -/mice using PPARα agonists. We did not make a statistical comparison of the expression levels of CCL2 in the control group and the model group in immunofluorescence staining, the results of detecting CCL2 and PPARα protein expression of the left ventricular outflow tract tissues showed that the expression of the two proteins have no significant difference between the control group and the model group, we believe that there are some differences of data from cell model and mice model are reasonable, because Apoe -/mice are an animal model of spontaneous atherosclerosis, Apoe -/mice develop atherosclerotic plaques from 2 months of age, with or without a high-fat diet, the high-fat diet is only an accelerator to the plaque, the aortic tissue we deteced comes from 5-month-old Apoe -/mice, which had formed stable plaques with or without a high-fat diet, that is the reason that we did not detect expressional differences of CCL2 and PPARα between the control group and the model group.
While data from cell model (oxLDL for 24 hrs) showed induction of CCL2 and PPARα because of normal cells without any treatment as the control group, CCL2 and PPARα expressional level were induced by ox-LDL. We have examined the PPARα expression among these animal models, please check. Figure 6, the CCL2 expression level should be included in parallel with the effect of PPARα on proliferation, migration and angiogenesis. The control group (without oxLDL) should also be included.

9.
Answer 9: Dear reviewer, thanks for your professional advice firstly. We have detected the CCL2 expressional level after adding PPARα agonist to the stable CCL2 knockdown HUVECs, in order to investigate whether apoptosis, proliferation,