Arterial stiffness and cardiac dysfunction in Hutchinson–Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase

The findings show that increased lysyl oxidase abundance is causal for the elevated arterial stiffness present in the arteries of Hutchinson–Gilford Progeria Syndrome mice. Pharmacologic inhibition of lysyl oxidase improves cardiac dysfunction and restores arterial compliance.

For a brief overview, this manuscript was previously reviewed and accordingly revised for nonalliance journal. The authors then transferred the previous review comments, the editor's decision letters, their point-by-point rebuttal and a revised manuscript to Life Science Alliance (LSA). After a thorough review for the manuscript and the peer-review material, the manuscript was deemed to be appropriate for LSA, provided an arbitrator (referee) signs of on the revised version.
The referee's comments are copied below.
As you will note, the referee is overall enthusiastic about the manuscript. They have raised some points (cardiac dysfunction markers and miR145 loss of function analysis) that we encourage you to address only if the data is readily available. If the data is not readily available or attainable, we suggest you tone down the respective conclusions and address these issues in Discussion. The referee's request asking for better description of the RNA analysis (pt 3) in a publicly available database should be addressed.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. In this report, aortic stiffness was observed and characterized in HGPS and ageing mice at functional, morphological and molecular levels. Lox activation was identified as an early event and its contribution to aortic stiffening was tested using a specific Lox inhibitor. The mechanistic analysis for Lox activation was performed by transcriptome profiling which led to the finding of miR-145. While the role of miR-145 in Lox regulation was tested in SMCs, its involvement appears to be specifically limited to HGPS model rather than natural ageing.
The observation of early onset of aortic stiffening and the characterization of the underlying mechanisms in HGPS mice contributed new insights to the pathophysiology and mechanism of cardiovascular ageing due to LaminA mutation. The finding of Lox induction and miR-145 mediated regulation of Lox expression have potential value in mechanistic understanding, diagnosis and therapy.
1. The study provided comprehensive analysis of aortic stiffening at different time points and sex groups. The temporal correlation between Lox induction and ECM remodeling associated with aortic physiology are well performed and supported. However, the authors only tested the expression in aortic tissue, but showed Lox inhibition conferred significant improvement in cardiac stiffness as well. Yet, Lox expression and activity in cardiac tissue, along with histological measurement were not provided. Other markers of cardiac dysfunction, including marker genes should be analyzed to support the functional improvement in treated mouse hearts. Adding these data would strengthen the conclusions and impact of the finding.
2. mR145 was identified as a conserved regulator for LOX through correlated gene expression profile and gain of function analysis in culture. This is an important finding of this report, and the conclusion should be better substantiated by a loss of function analysis using antagomer for miR145 and the utilize a reporter assay to support the direct regulatory mechanism.
3.Authors need to provide better description of the RNA analysis, how many samples were sequenced, how reproducible of each sample in global gene expression patterns (illustrated by PCA) and how many total DEG transcripts were identified. What is the outcome of IPA analysis in terms of cellular processes and pathways affected in the HGPS tissues. In short, more comprehensive transcriptome data analysis would add much value and impact to this report as well. Thank you for your positive comments and those of the arbitrator. We were delighted to hear that you look forward to publishing our paper in Life Science Alliance.
We have followed your advice and have added data requested by the arbitrator when available or toned down the respective conclusions and addressed the limitations in the Discussion. We have provided a better description of the RNA analysis and submitted the primary data to a public repository as per your request. All of these points are specifically addressed below.

Arbitrator Comments
1. Lox expression and activity in cardiac tissue, along with histological measurement were not provided. Other markers of cardiac dysfunction, including marker genes should be analyzed to support the functional improvement in treated mouse hearts. REPLY: The COVID-19 pandemic has significantly limited our work on the heart. As this data is not readily available, we toned down our conclusions and stated limitations and the need for additional work in the Discussion (lines 343-348).
2. mR145 was identified as a conserved regulator for LOX through correlated gene expression profile and gain of function analysis in culture. This is an important finding of this report, and the conclusion should be better substantiated by a loss of function analysis using antagomer for miR145 and the utilize a reporter assay to support the direct regulatory mechanism.
REPLY: In fact, we previously showed that a miR-145 antagomer regulates LOX mRNA levels in smooth muscle cells and that the direction of its effect is complimentary to that of ectopic miR-145 expression. But we thank the arbitrator for making us realize that those prior results were not stated in this manuscript. We now describe and cite that prior study with LOX and the miR145 antagomer (Discussion: lines 359-362).
We do not have available data from a reporter assay, so have modified the Discussion to indicate that such a result could further strengthen the connection between miR-45 and LOX mRNA abundance (Discussion; lines 365-367).
3. Authors need to provide better description of the RNA analysis, how many samples were sequenced, how reproducible of each sample in global gene expression patterns (illustrated by PCA) and how many total DEG transcripts were identified. What is the outcome of IPA analysis in terms of cellular processes and pathways affected in the HGPS tissues. In short, more comprehensive transcriptome data analysis would add much value and impact to this report as well.
REPLY: As requested, we have provided a better description of the RNA analysis. We added the requested information about sample size to Material and Methods (line 488) and the approximate number of DEGs to Results (line 253). We have also provided the requested PCA and pathway (gene ontology) analysis (new Figure S13A . Finally, we have deposited the RNASeq files into GEO and provide the accession number (lines 500 and 535).
We hope the new data, the more accurate description of our previous work with a miR-145 antagomer, and the modifications we have made to the text make our study acceptable for publication in Life Science Alliance.