Hyaluronan Regulates Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Roy et al. describes the role of Hyaluronan (HA) in vascular calcification, using both in vitro and in vivo mouse models. The paper is well written and addresses a very interesting topic. Furthermore, the authors attempt to explore the specific roles of different HA synthases (HASes) in this process.

However, there are several points that should be addressed to strengthen the manuscript:

1. To better visualize the pericellular HA matrix, the authors might consider using a particle exclusion assay (e.g., using fixed red blood cells). Do the authors believe this alternative approach could further enhance or validate their current findings?

2. It is not clear which HAS isoenzyme is most prominently expressed in cells at the various time-points investigated following OM treatment. The authors should provide a re-analysis of their qPCR data to show the relative expression of the three HASes (e.g., by setting the lowest expressed isoenzyme to 1 and normalizing the others accordingly).

3. To more clearly distinguish the specific roles of the different synthases, did the authors consider silencing HAS1 and HAS2 (e.g., via siRNA or shRNA)? This could provide more definitive evidence of their individual contributions. Conversely, do the authors have the possibility to overexpress HAS3 to investigate a potential inhibition of osteogenic differentiation? Additionally, the authors should clearly report the efficiency of HAS1/HAS2 overexpression and HAS3 silencing compared to untreated controls (e.g., via qPCR or Western Blot).

4. It is well known that HAS3 synthesizes low molecular weight (LMW) HA, whereas HAS1 and HAS2 produce higher mass chains. Have the authors considered adding exogenous HA of different molecular weights to the Osteogenic Medium (OM) to support their findings regarding the protective or pro-calcific roles of HA?

5. In my opinion, the Discussion section should include a paragraph explaining the different sizes of HA produced by the three synthases. It would be beneficial to discuss the putative role of LMW-HA produced by HAS3 versus the small fragments generated by hyaluronidases, as well as the potential inhibitory role of high molecular mass (HMW) HA produced by HAS1 and HAS2.

6. The graphs reporting the quantifications in all the figures are currently too small and difficult to read. I suggest increasing the font size and the overall scale of these panels for better clarity.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors examine the effects of recombinant IL6 or TGFß1 treatment on the calcification of vascular smooth muscle cells in osteogenic medium and observe that changes in the expression of osteogenic markers coinciding with alterations in hyaluronan as well as with enzymes involved in its synthesis or degradation.

Overall, the manuscript is well written, the data well presented and the conclusions carefully drawn. The discussion could be extended towards potential mechanism and translational aspects in this regard. Since the majority of the experiments are in vitro, relying on stimulation experiments without a time and a dose curve and further insights into potential signaling events, these limitations should be discussed more extensively. Histological validation experiments in a mouse model of vitamin K deficiency confirm the main observations, which still does not definitely prove causality.

 

Specific comments:

1. The limitations of the study regarding causality between HAS1 and HAS2 downregulation and HA deposition and calcification are discussed by the others. Can the authors also speculate and discuss what alters the HA synthesis and breakdown pathway? Since direct evidence that hyaluronan regulates VSMC osteogenic differentiation is not presented, the title should be modified and reflect the data shown.
2. Is this pathway specific for arterial media calcification and patients with chronic kidney disease and diabetes, as suggested by the Introduction, or independent on the underlying pathology?
3. In the methods part, I noted that HAS1 and HAS2 were overexpressed using plasmid transfection, HAS3 downregulated using siRNA. Why? Although is says short-term transfection, images (e.g. in Figure 8) are until from day 21 in culture. Please indicate (in the figure) at which time point the plasmid/siRNA transfection was performed.
4. Could the absence of effects seen after HAS3 downregulation be a technical or methological problem?
5. Results shown in Figure 1 are largely an establishment of the culture conditions, i.e. calcification of SMCs under osteogenic medium. The group is working on vascular calficiation for years. Therefore, an introductory sentence here but also some more details in the Methods would help how the experimental conditions here differ from previous, published ones (including other groups).
6. Please indicate what the data points in the graphs refer to and whether they are biological or experimenal replicates or microscope images. In the majority of cases, n=3 data points were variable are shown, which is the absolute minimun to perform any statistical analysis.
7. Alizarin Red detects established calcification, RUNX2 is a transcription factor regulated in earlier stages of osteoblastic differentiation. Maybe this could also explain some of the discrepant results?
8. Figure 4: I am not sure how intra- and extracellular HA was distinguished on the cellular level, as no clear cell borders can be seen on the images. Should it not also be visible in cultivated cells?
9. Figure 5E and 5F: why were two different fluorescence labels used to visualize HAS3 (upper and lower panels in F?) Which of them was quantified in E. It is not explained in the legend.
10. How IL6 and TGFß1 upregulate VC in SMCs is not further examined in the study and remains open. Since both cytokines signal via different receptors and pathways, possibilities should be at least discussed, also in terms of potential strategies to halt calcification in patients.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf