Long Non-Coding RNA MALAT1 Regulates HMOX1 in Sickle Cell Disease-Associated Pulmonary Hypertension

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an investigation into the role of the long non-coding RNA MALAT1 in the pathogenesis of pulmonary hypertension associated with sickle cell disease. The authors employ a combination of in vivo mouse models, in vitro cellular assays, and molecular analyses to elucidate the MALAT1-HMOX1 axis as a potential therapeutic target. The study addresses an important gap in understanding the regulatory mechanisms of endothelial dysfunction in SCD-PH and offers novel insights with potential clinical implications.

1. Introduction would benefit from a more explicit statement of the current gaps in knowledge regarding the molecular mechanisms regulating endothelial dysfunction in SCD-PH. For example, while MALAT1 is introduced as a factor of interest, its specific known functions in vascular pathology prior to this study are not thoroughly described.
2. The rationale for focusing on the MALAT1-HMOX1 axis is generally implied through the mention of prior findings of MALAT1 upregulation in SCD models. Explicitly stating the hypothesis and how this axis in the introduction section might represent a novel regulatory pathway would strengthen the narrative.
3. The direct regulation of HMOX1 by MALAT1 remains to be more conclusively demonstrated. Consider including experiments that show MALAT1 binding or interaction with HMOX1 mRNA or promoter regions
4. All abbreviations are well-defined; consider including a table of abbreviations for quick reference.
5. Discuss the feasibility and challenges related to targeting MALAT1 therapeutically in humans.

Author Response

Reviewer 1

Comment #1. Introduction would benefit from a more explicit statement of the current gaps in knowledge regarding the molecular mechanisms regulating endothelial dysfunction in SCD-PH. For example, while MALAT1 is introduced as a factor of interest, its specific known functions in vascular pathology prior to this study are not thoroughly described.

Response #1.1. We appreciate the reviewer’s insightful comment. The Introduction has been revised to provide a clearer statement of the current knowledge gaps regarding the molecular mechanisms regulating endothelial dysfunction in SCD-associated pulmonary hypertension (SCD-PH). We have expanded our discussion of MALAT1’s previously reported roles in vascular pathology, including its involvement in endothelial cell activation, inflammatory signaling, and vascular remodeling in other disease contexts.

 

Comment #1.2. The rationale for focusing on the MALAT1-HMOX1 axis is generally implied through the mention of prior findings of MALAT1 upregulation in SCD models. Explicitly stating the hypothesis and how this axis in the introduction section might represent a novel regulatory pathway would strengthen the narrative.

Response #1.2. We thank the reviewer for this constructive suggestion. In the revised manuscript, we have clarified the rationale for focusing on the MALAT1–HMOX1 axis by explicitly stating our hypothesis in the Introduction. We now emphasize that, based on prior evidence of MALAT1 upregulation in SCD models and its known regulatory roles in endothelial cell function, we hypothesized that MALAT1 contributes to endothelial dysfunction in SCD-PH through regulation of HMOX1 expression.

 

Comment #1.3. The direct regulation of HMOX1 by MALAT1 remains to be more conclusively demonstrated. Consider including experiments that show MALAT1 binding or interaction with HMOX1 mRNA or promoter regions.

Response #1.3. We appreciate the reviewer’s thoughtful comment. Currently, our ongoing studies are focused on investigating the potential indirect regulation of HMOX1 by MALAT1, possibly through intermediary transcriptional or post-transcriptional mechanisms. We have clarified this point in the revised Discussion section for future research.

 

Comment #1.4. All abbreviations are well-defined; consider including a table of abbreviations for quick reference.

Response #1.4. We thank the reviewer for this helpful suggestion. To improve readability and accessibility, we have added a table summarizing all abbreviations used in the manuscript

 

Comment #1.5. Discuss the feasibility and challenges related to targeting MALAT1 therapeutically in humans.

Response #1.5. We appreciate the reviewer’s insightful comment. In the revised Discussion, we have added a section addressing the feasibility and challenges of targeting MALAT1 therapeutically in humans.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Pathophysiology of sickle cell disease (SCD) includes pulmonary hypertension (PH) as a co-morbidity.

Here, Sueblinvong et al provide an unbiased assessment of Long non-coding RNAs dysregulation in PH using mice with SCD and normal controls. Thousands of lncRNAs were found to be differentially expressed, and authors moved on to validate the top hits.

Somehow, by analysing only the top hits, authors give the impression to analyse the very valuable array data only at the surface. What about providing gene ontology analysis ? Ingenuity pathway analysis? and KEGG ? Microarray data yield so much more that simply the top hits that a more thoughtful bioinformatics analysis would be warranted.

Among those, MALAT1 and HMOX1, overexpressed in SCD mice, were further tested in in vitro in Human pulmonary artery endothelial cells HPAECs, and their expression was shown to be dependent on haemin added to the medium (haemin being a circulating heme degradation producy in SCD.

Overall, results support a role for a MALAT1 - HMOX1 signalling in SCD pointing at them as putative therapeutic targets.

Line 89 : please state the DMSO concentration in the cell culture medium

Proof that AdGFP and AdMALAT1 administration leads to protein overexpression must be provided.

There is some confusion between the notion of lncRNA and mRNA. As the Arrystar™ lncRNA array provides a profile of both long non-coding RNAs (lncRNAs) AND the entire set of protein-coding mRNAs, authors should be more precise on what is the primary observation. Do they observed overexpressed lncRNAs for MALAT1 - HMOX1 ? Or overexpressed mRNA ? Or both ?

Provision of a supplementary table with all the up- down- regulated entries would be beneficial. Or deposition into a public database. The statement at line 400 does not help to locate the microarray raw data.

Please add a table reporting primers used for qPCR.

Author Response

Reviewer 2

Comment #2.1. Here, Sueblinvong et al provide an unbiased assessment of Long non-coding RNAs dysregulation in PH using mice with SCD and normal controls. Thousands of lncRNAs were found to be differentially expressed, and authors moved on to validate the top hits. Somehow, by analysing only the top hits, authors give the impression to analyse the very valuable array data only at the surface. What about providing gene ontology analysis ? Ingenuity pathway analysis? and KEGG ? Microarray data yield so much more that simply the top hits that a more thoughtful bioinformatics analysis would be warranted.

Response #2.1. We appreciate the reviewer’s thoughtful comment regarding the depth of bioinformatics analysis. In the current manuscript, we focused on validating the top differentially expressed lncRNAs to provide a clear and concise mechanistic story. We acknowledge that microarray data offer broader insights beyond the top hits, and we are currently preparing a separate manuscript that includes comprehensive analyses of the lncRNA array data, such as gene ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and KEGG pathway enrichment. We have added this point to the discussion on future work.

 

Comment #2.2. Line 89 : please state the DMSO concentration in the cell culture medium.

Response #2.2. We thank the reviewer for pointing this out. The DMSO concentration used in the cell culture medium was 0.1%, and this has now been explicitly stated in the Methods section.

 

Comment #2.3. Proof that AdGFP and AdMALAT1 administration leads to protein overexpression must be provided.

Response #2.3. We thank the reviewer for this important suggestion. Although we confirmed significant increases in ET-1 (Figure 3D) , VCAM1 (Figure 3E), and HMOX1 (Figure 5A) mRNA levels upon MALAT1 overexpression, we have not yet directly examined the corresponding protein expression changes. We plan to perform Western blot analyses in future studies to further validate the regulation of MALAT1-associated proteins following AdMALAT1 administration.

 

Comment #2.4. There is some confusion between the notion of lncRNA and mRNA. As the Arrystar™ lncRNA array provides a profile of both long non-coding RNAs (lncRNAs) AND the entire set of protein-coding mRNAs, authors should be more precise on what is the primary observation. Do they observed overexpressed lncRNAs for MALAT1 - HMOX1 ? Or overexpressed mRNA ? Or both ?

Response #2.4. We appreciate the reviewer’s helpful comment and the opportunity to clarify this point. In our analysis, both the long non-coding RNA MALAT1 and the HMOX1 mRNA were found to be increased in the SS group compared with controls.

 

Comment #2.5. Provision of a supplementary table with all the up- down- regulated entries would be beneficial. Or deposition into a public database. The statement at line 400 does not help to locate the microarray raw data.

Response #2.5. We thank the reviewer for this suggestion. To improve transparency and accessibility, we have now provided a Supplementary Table S1 listing all up- and down-regulated lncRNAs identified in our microarray analysis.

 

Comment #2.6. Please add a table reporting primers used for qPCR.

Response #2.6. We thank the reviewer for this helpful suggestion. A new Table 1 has been added in the methods listing all qRT-PCR primers used, including their sequences.

Reviewer 3 Report

Comments and Suggestions for Authors

 Dear Dr. Authors, Thank you for your hard work and for the well-presented article. The work is well-written, academically sound, and engagingly readable. I have some suggestions.

Abstract:
1) It is desirable to improve the results section in the abstract; 
Introduction;
2) Line 54- I suggest that authors add suitable references;
3)Line 71- I suggest adding two or more suitable references;
4) I suggest that authors may add 2 or more sentences regarding the MALAT1 role/implication in disease;
Methods:
4) Line  150:  regarding the qRT-PCR analysis, add the system's name, program, etc.  Authors allow the reference  "30" https://journals.physiology.org/doi/full/10.1152/ajplung.00195.2011 
The used system was the same, the run program was the same?
Results:
5) "Figure 1. Hemolysis induces ALAT1 levels in vivo and in vitro"  and  "Figure 3. MALAT1 overexpression attenuates RV systolic pressure, RVH, vascular remodeling, and endothelial dysfunction markers ET-1 and VCAM1 gene expressions"  in both cases. In the text, the explanation can be extended (become informative); I suggest that the authors further explain;
6) 3.4. HMOX1 protects endothelial function in SCD-PH; this must be informative based on the results. This is possible based on the obtained results;
Discussion part:
7) I suggest to authors update regulatory networks involving various types of non-coding RNAs's paragraph add current/new stiudies; It would be ineterseting  to update in context  of the  diagnostic/therapeutic; 
8) I suggest to authirs add future perspectives according their view (based on conducted results);

Thank You

Best Regards

Author Response

Reviewer #3

Comment #3.1. It is desirable to improve the results section in the abstract. 

Response #3.1. We appreciate the reviewer’s suggestion. The Results section of the abstract has been revised to provide a clearer and more concise summary of our key findings, emphasizing the expression of MALAT1 and HMOX1 and their potential roles in endothelial dysfunction in SCD-PH.

 

Comment #3.2. Line 54- I suggest that authors add suitable references. Line 71- I suggest adding two or more suitable references.

Response #3.2. We thank the reviewer for the suggestion. Appropriate references have been added to support the statements on lines 54 and 71, providing additional context and supporting evidence for the points made in the manuscript.

 

1. Kato GJ, McGowan V, Machado RF, Little JA, Taylor 6th J, Morris JR, Nichols JS, Wang X, Poljakovic M, Morris Jr SM, Gladwin MT. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood. 2006;107(6):2279-2285.
2. Tai YY, Yu Q, Tang Y, Sun W, Kelly NJ, Okawa S, Zhao J, Schwantes-An TH, Lacoux C, Torrino S, Al Aaraj Y, El Khoury W, Negi V, Liu M, Corey CG, Belmonte F, Vargas SO, Schwartz B, Bhat B, Chau BN, Karnes JH, Satoh T, Barndt RJ, Wu H, Parikh VN, Wang J, Zhang Y, McNamara D, Li G, Speyer G, Wang B, Shiva S, Kaufman B, Kim S, Gomez D, Mari B, Cho MH, Boueiz A, Pauciulo MW, Southgate L, Trembath RC, Sitbon O, Humbert M, Graf S, Morrell NW, Rhodes CJ, Wilkins MR, Nouraie M, Nichols WC, Desai AA, Bertero T, Chan SY. Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension. Sci Transl Med. 2024;16(729):eadd2029.
3. Zahid KR, Raza U, Chen J, Raj UJ, Gou D. Pathobiology of pulmonary artery hypertension: role of long non-coding RNAs. Cardiovasc Res. 2020;116(12):1937-1947.

 

Comment #3.3. I suggest that authors may add 2 or more sentences regarding the MALAT1 role/implication in disease.

Response #3.3. We appreciate the reviewer’s suggestion. We have added two sentences in the Introduction/Discussion highlighting the roles of MALAT1 in disease, including its involvement in endothelial activation, inflammation, and vascular remodeling, as well as its potential contribution to the pathogenesis of vascular complications in SCD. (Please see Response #1.1.)

 

Comment #3.4. Line 150:  regarding the qRT-PCR analysis, add the system's name, program, etc.  Authors allow the reference "30" https://journals.physiology.org/doi/full/10.1152/ajplung.00195.2011 The used system was the same, the run program was the same?

Response #3.4. We thank the reviewer for this comment. The qRT-PCR analysis was performed using the same system and cycling program as described in reference 30.

 

Comment #3.5. "Figure 1. Hemolysis induces MALAT1 levels in vivo and in vitro" and  "Figure 3. MALAT1 overexpression attenuates RV systolic pressure, RVH, vascular remodeling, and endothelial dysfunction markers ET-1 and VCAM1 gene expressions” in both cases. In the text, the explanation can be extended (become informative); I suggest that the authors further explain.

Response #3.5. We appreciate the reviewer’s suggestion. The figure legends for Figures 1 and 3 have been revised to provide more detailed explanations of the experimental design, key findings, and their significance.

 

Comment #3.6. 3.4. HMOX1 protects endothelial function in SCD-PH; this must be informative based on the results. This is possible based on the obtained results.

Response #3.6. We thank the reviewer for this insightful comment. Based on our results, we have revised the relevant section to clearly state that HMOX1 appears to protect endothelial function in SCD-PH. Specifically, our data show that MALAT1 knockdown reduces HMOX1 levels and increases endothelial dysfunction markers ET-1 and VCAM1, supporting a protective role for HMOX1 in maintaining endothelial homeostasis in this context.

 

Comment #3.7. I suggest to authors update regulatory networks involving various types of non-coding RNAs's paragraph add current/new studies; It would be interesting to update in context of the diagnostic/therapeutic; 

Response #3.7. We appreciate the reviewer’s insightful suggestion. In the revised manuscript, we have expanded the section discussing the regulatory networks involving various types of non-coding RNAs (ncRNAs), incorporating recent studies to provide a more comprehensive overview.

 

Comment #3.8. I suggest to authors add future perspectives according to their view (based on conducted results).

Response #3.8. We thank the reviewer for this valuable suggestion. In the revised manuscript, we have added outlining future perspectives based on our results.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor, dear authors:

Relatively to the paragraph: "2.3. Assessment of PH in MALAT1-overpressing sickle cell mice" I requested as "mandatory" to provide evidence for adenoviral-mediated expression of GFP (in control mice) and MALAT1, in AdMALAT1-treated mice.

You must convene with me that, as long as the occurrence of adenoviral-mediated expression of a protein is not proven, any furter experimental result is , at least, questionable. (My previous Comment #2.3.)

This is the reason why I do not consider the manuscript as complete at this stage.

 

Author Response

Comment #2.3. Proof that AdGFP and AdMALAT1 administration leads to protein overexpression must be provided. 

Response #2.3. We thank the reviewer for this important comment. In the revised manuscript, we performed Western blot analyses demonstrating that adenovirus-mediated MALAT1 overexpression increased Hmox1 protein expression in the mouse lungs. Figure 5A and Supplementary Figure have been added with Western blot image. 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

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Author Response

Comment #2.3. Proof that AdGFP and AdMALAT1 administration leads to protein overexpression must be provided.

Response #2.3. We thank the reviewer for this important comment. In the revised manuscript, we included Supplementary Figure S1A-B which show the administration of AdMALAT1 led to a significant upregulation of Malat1 in the mouse lungs and transfection of AdMALAT1 led to a significant increase in Malat1 in HAPECs.