Ending Restenosis: Inhibition of Vascular Smooth Muscle Cell Proliferation by cAMP

Round 1

Reviewer 1 Report

This is a thorough and well written review by a group that has been working on this theme. It goes through the history of the subject to the most recent data in the literature. It provides a large amount of information on the subject with adequate bibliography. The 3 figures are valuable additions that help one to follow the complex signaling pathways described in the text. I feel the paper is of high enough quality for publication with a few minor corrections:

- Abbreviation should defined in the abstract (Skp2, SRF, TEAD, ..) and figure legends (CDKIs, …).

- Figures 1-3 should be indicated in the text. They could be presented in color to increase their impact.

- Lane 300 : Ref 135 is not adequate

- Figure 2. Actin monomer should be mentioned in the cartoon. In addition, it is difficult to see the distinct effect of MKL1/2 bound actin monomer with MKL1/2 unbound actin on SRF gene expression. Yet, perhaps the effect of LATS and MST on YAP/TAZ  could be better presented in the figure.

- Figure 3 legend. I would suggest to describe the effect of MSK1/2.

Author Response

REVIEWER 1

Comment 1: Abbreviation should defined in the abstract (Skp2, SRF, TEAD, ..) and figure legends (CDKIs, …).

Response 1: All abbreviation in the abstract and figure legends have now been defined.

Comment 2: Figures 1-3 should be indicated in the text. They could be presented in colour to increase their impact.

Response 2: Figures 1 to 3 have now been indicated in the text

Comment 3: Lane 300: Ref 135 is not adequate

Response 3: This reference has now been corrected.

Comment 4: Figure 2. Actin monomer should be mentioned in the cartoon. In addition, it is difficult to see the distinct effect of MKL1/2 bound actin monomer with MKL1/2 unbound actin on SRF gene expression. Yet, perhaps the effect of LATS and MST on YAP/TAZ  could be better presented in the figure.

Response 4: Actin monomer has now been indicated in the diagram. We hope that the more clearly labelling of actin monomer will help make the effects of actin monomer on MKL1/2 clearer. We have also added an arrow to indicate that actin monomer induces MKL nuclear export, which is described in the legend.

Comment 4:  Figure 3 legend. I would suggest describing the effect of MSK1/2.

Response 4: We have added a mention of the role of MSK1/2 to the legend.

Reviewer 2 Report

This is a well-constructed and relatively comprehensive review of the impact of cyclic AMP and its signals on vascular restenosis.  Several concerns do exist though:

The authors failed to include discussion of VASP, an important kinase-driven downstream effector of cAMP (and cGMP) in vascular smooth muscle (VSM) physiology and pathology including restenosis and neointimal development. 

There is a lack of discussion of cyclic nucleotide-stimulating activators and stimulators (as upstream cAMP (and cGMP) inducers) and their biological and clinical significance?  Given the clinical importance of this family of activators and stimulators in relation to cAMP signaling and biology, it would be recommended to include at least a brief discussion of this impact on these parameters.

There is inadequate discussion about cAMP-specific phosphodiesterases (PDEs), which act to balance cAMP signaling, which is curious given their broad clinical significance. 

When discussing VSM cell proliferation and VSM growth, increases can lead to neointimal formation and restenosis following primary occlusion; however, decreases in the medial wall can lead to mural thinning and aneurysm formation.  For a comprehensive review, this article should include discussion about the permutations of reduced VSM cell proliferation and growth and pathologies (like aneurysm development) associated with that.

Given the promiscuity of cyclic nucleotide and serine/threonine kinase signaling, discussion of alternate targets/effectors of cAMP should include PKG and PKG-mediated events.

With emphasis on vascular restenosis, it would be helpful for the reader if schematics were shown (possibly including photomicrographs) depicting the impact of cAMP on restenosis and/or neointimal formation.  The only figures shown are signaling schematics.

A comprehensive listing of references is provided, yet many of the references are decades old, and this includes those cited for relevant timely data rather than just for those given to provide historical perspectives.  For these issues, it would be advised to update those references to inform the reader of the current state of knowledge of those issues.

Author Response

We thank the reviewer for the helpful and constructive suggestions to improve our review. We have made several additions and amendments as suggested. These include:

1          We have added a discussion of the important role of VASP phosphorylation as a PKA/PGK substrate

2          We are not clear what the reviewer means when suggestion additional discussion of cyclic-nucleotide-stimulating activators and stimulators? We agree that inclusion of a discussion of upstream physiological signals that stimulate cAMP is important and we have included a detailed discussion of the importance of adenosine and prostacyclin as important vasoactive GPCR agonist that elevate cAMP and modulate VSMC proliferation

3          We agree that our original manuscript lacked sufficient discussion of the importance of PDEs. We have now added more discussion of the importance of PDE enzymes.

4          We agree that various vascular pathologies are associated with medial thinning and aneurysm formation. However, this is often as a result of VSMC senescence or apoptotic cell death. This area justifies an entire review in it own right and we feel that inclusion of a discussion of these mechanisms here would detract from the focus on proliferation and cAMP. There is also not a lot of literature on the role played by cAMP signalling in medial thinning and aneurysm formation. Therefore, we would prefer not to include a discussion of these aspects of vascular disease in the current article.

5          We agree that we should mention the research suggesting that cAMP can mediate effects via PKG. We have now included this in the revised manuscript.

6          We have added numerous new references to ensure that we cite the most recent research.

Reviewer 3 Report

This is a rather comprehensive review about the mechanisms of cAMP inhibition of vascular smooth muscle proliferation. The title is catchy, maybe somewhat too catchy considering the standing of the field and how far we actually are from solving the problem of restenosis, but certainly helps addressing the relevance of the field and may attract readers. I find the review well written and timely, since there are not many similar reviews lately. Although much of the reviewed work is now relatively old, it is nicely set in context with new research and I find it useful that it is all included. I have mainly minor suggestions for improvement.

Specific comments:

Line 79: “However, elevation of cAMP paradoxically increases proliferation in embryonic stem cells [21], epithelial cells [22], endothelial cells [23,24] and even VSMC under some conditions [25].” It would seem reasonable to include here also several endocrine tissues/cells, where cAMP is a trophic signal. And why is this paradoxical?

Line 93 an several other occurences: “adenylate cyclase” should be written “adenylyl cyclase” to correctly reflect the chemistry of the reaction and recent agreement on the nomenclature.

Lines 98-118, regarding the discussion about adenosine and adenosine receptors: Is there any evidence for effects of caffeine in this context? If so, would be nice to include.

Line 239 “6. The role of cAMP sensors”. Maybe say cAMP effectors, to avoid confusion with e.g. FRET-based cAMP sensors used as tools today?

Line 292: “…selective EPAC activation alone using 8-(4-Chlorophenylthio)adenosine- 3', 5'- cyclic monophosphate (8-CPT-cAMP) …”. Do you mean 8-CPT-2′-O-Me-cAMP? Please be precise, as 8-CPT-cAMP is a potent stimulator of both PKA, PKG and EPAC, as well as a PDE5 inhibitor.

Lines 298 and 324: “8-CPT-cAMP”: Same as above

Lines 461-462: In Figure 2, the direction of the arrows indicating polymerization and depolymerization appears opposite of what I expected. Please check.

Line 559-579, The section “9. Pharmacological and clinical implications”: This part is relatively brief and could benefit from some expansion. E.g., does the knowledge have any implications for current pharmacological treatment? What, if any, are the effects of non-selective beta-blockers on VSMC proliferation? What about beta-2 agonists? Effects of caffeine? PDE inhibitors? Or do the authors want to limit the discussion to “Future pharmacological and clinical implications” (if so, change the heading)

General comments, language etc.:

Please make sure that all abbreviations are defined on first use.

Although not many, there are some typos and minor mistakes throughout.

E.g.:

Line 20: Change “for” to “of”?

Line 90: Delete “with”

Line 159: indicated --> indicating

Line 230: Rb0 --> Rb

Line 271: either “smooth muscle cell” or “SMC”, not both

Line 328: hat --> that

Line 369: “the rate” is duplicated

Line 381: restiction --> restriction

Line 445: induces --> induce

Line 509: import --> important

Line 523: “concentrations” is lacking

Line 589. “insult of injury”?

References: The reference format is inconsistent, especially regarding journal names and/or abbreviations, as well as capitalisation, and there are several typos in the titles. Please check carefully.

Author Response

REVIEWER 3

Comment 1: Line 79: “However, elevation of cAMP paradoxically increases proliferation in embryonic stem cells [21], epithelial cells [22], endothelial cells [23,24] and even VSMC under some conditions [25].” It would seem reasonable to include here also several endocrine tissues/cells, where cAMP is a trophic signal. And why is this paradoxical?

Response 1: We have added thyrocytes to this sentence.

Comment 2: Line 93 an several other occurences: “adenylate cyclase” should be written “adenylyl cyclase” to correctly reflect the chemistry of the reaction and recent agreement on the nomenclature.

Response 2: We have corrected all instances of adenylate cyclase to “adenylyl cyclase” throughout.

Comment 3: Lines 98-118, regarding the discussion about adenosine and adenosine receptors: Is there any evidence for effects of caffeine in this context? If so, would be nice to include.

Response 3: We agree that the effects of caffeine as an adenosine receptor antagonist are interesting and this suggest that caffeine may play a role in regulating VSMC proliferation. However, we were unable to find ant published research demonstrating clear effects of caffeine on VSMC proliferation via effects on adenosine receptors.

Comment 4: Line 239 “6. The role of cAMP sensors”. Maybe say cAMP effectors, to avoid confusion with e.g. FRET-based cAMP sensors used as tools today?

Response 4: We have changed the title of this section to The role of cAMP effectors as suggested.

Comment 5: Line 292: “…selective EPAC activation alone using 8-(4-Chlorophenylthio)adenosine- 3', 5'- cyclic monophosphate (8-CPT-cAMP) …”. Do you mean 8-CPT-2′-O-Me-cAMP? Please be precise, as 8-CPT-cAMP is a potent stimulator of both PKA, PKG and EPAC, as well as a PDE5 inhibitor.

Response 5: We apologise for this error, which we have now corrected.

Comment 6: Lines 461-462: In Figure 2, the direction of the arrows indicating polymerization and depolymerization appears opposite of what I expected. Please check.

 Response 6: The reviewer is correct that these arrows were the wrong way around. We have now corrected them.

Comment 7: Line 559-579, The section “9. Pharmacological and clinical implications”: This part is relatively brief and could benefit from some expansion. E.g., does the knowledge have any implications for current pharmacological treatment? What, if any, are the effects of non-selective beta-blockers on VSMC proliferation? What about beta-2 agonists? Effects of caffeine? PDE inhibitors? Or do the authors want to limit the discussion to “Future pharmacological and clinical implications” (if so, change the heading)

Response 7: We have now changed the heading of this section to “Future pharmacological and clinical implications” as suggested.

General comments:

All abbreviations have now been defined and the detailed typographical errors corrected.