Amyloid Beta Peptides and Th1 Cytokines Modulate Human Brain Vascular Smooth Muscle Tonic Contractile Capacity In Vitro: Relevance to Alzheimer’s Disease?

Round 1

Reviewer 1 Report

Your research was adequately addressed and reported.

Author Response

We appreciate the reviewer's positive comments and for their time reviewing our submission. Thank you very much!

Reviewer 2 Report

This study introduces the relationships between the contractility of HBVSMC and multiple types of amyloid-ß peptide (Aß). The authors have insisted that the presence of Aß1-42 or Aß25-35, HBVSMC contractility could be induced while Aß1-40 could not affect the contractility of HBVSMC. In addition, the effect of various cytokines on HVBSMC contractility have been investigated. The writing is straightforward and the experimental/theoretical approaches of the presented research make it attracted to the readership of the Pathophysiology; however, major revision of the manuscript is necessary prior to publication.

 

Comments:

1. Have the authors investigated the effect of Tau proteins or various ROS on the contractility of HBVSMC instead Aß?
2. The experimental results (Figure 1) showed that Aß1-42 peptide could decrease HBVSMC contractility, however, except the third day, both 5 and 10 uM of Aß1-42 may not be able to affect the contractility of HBVSMC. The error bars looked like overlapped. Please offer more clear discussion about this.
3. The contractility of HBVSMC in the presence of Aß25-35 showed lower than the samples without Aß25-35 (Figure 3). Please describe the results more clearly in the manuscript.
4. Based on Figure 1, 2, and 3, the control samples showed different influence on the contractility of HBVSMC. To me, control samples should present very similar results. Please explain this in the manuscript clearly.
5. The control samples used for the experiments with inflammatory cytokines should present similar results, however, they do not. Please provide explanation why the control samples showed different results in different experiments.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

The brains of Alzheimer’s disease patients are characterized by accumulation of amyloid beta (Ab)-containing extracellular plaques and enhanced release of pro-inflammatory cytokines in the brain. Both Aβ plaques and pro-inflammatory cytokines cause deleterious effects in brain cells. Brain vascular smooth muscle cells (BVSMC) are components of cerebral blood vessels and regulate cerebral blood flow. Thus, any negative effect on these cells can dysregulate cerebrovascular function.

  Authors aimed to study the effects of Aβ peptides on brain vascular smooth muscle cells (BVSMC) contractility. They observed that Aβ1-42 and IFN-g, TNFα, IL-1β decreased contractility in a cell line of hBVSMC.

Here is a list of the main concerns in the manuscript:

1. Abstract, Introduction and Results contain several concepts that are not addressed in the manuscript (cerebral autoregulation, cerebral blood flow, cerebral micro bleeds, iron deposition, neuroinflammation, microglial activation…). These sections should be re-written.
2. Overstatements:
   • (Lines 91-94) “We propose that impaired cerebral autoregulation and resulting micro bleeds and potentially increased Ab deposition in AD patients may be mediated by Ab peptides and elevated inflammatory cytokines with dysregulate brain vascular smooth muscle cell functioning.” Authors only showed that Aβ and some inflammatory cytokines affect contractility of a cell line hBVSMC.
   • (Lines 247-251) “Our data now suggest that inflammatory cytokines may not only mediate progression of disease by damaging microglia and neurons via amyloidogenicity as previously shown, but also directly affecting brain vascular smooth muscle cells responsible for maintenance of constant blood flow via cerebral autoregulation.” It seems that authors also tested the effects of inflammatory cytokines in microglia and neurons.
3. Results:
   • hBVSMC were treated with cytokines for six days. Are these cytokines stable in cell culture for that time of incubation?
   • A figure of collagen gels would be very useful for a better interpretation of the data.
   • Figure 3a is confusing, all lines are overlapped and significance is not convincible.
   • The quality of the Figure 4 is poor.
   • What is the rationale of combining IFN-g with TNF-α or TNF-α with IL-1β?
   • The effects of Aβ and cytokines on hBVSMC contractility are addressed by only one method. Further evidence is highly recommended.
4. Discussion:
   • Why different sizes of Aβ fragments cause opposite effects on hBVSMC contractility is not discussed.
   • It is not clear why authors focused the study on the effects of both Aβ or cytokines on hBVSMC contractility. Why these aspects of Alzheimer’s disease and not others?
   • Why the effect of cytokines on hBVSMC contractility is higher than Aβ is not discussed.
   • Several parts of the Discussion read as for an Introduction.
5. References:
   • References are not recent. 23/30 references were published more than 5 years ago.
   • Previous studies about Ab and VSMC contractility (Hald, 2016, J Biochem Eng; Vromman, 2012, Aging Cell) are not listed in References.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The revised manuscript has been improved description of the relationships between the contractility of HBVSMC and multiple types of Aβ. This manuscript has been improved compared to the previous version and attractive to readers of Pathophysiology.

Author Response

We appreciate the reviewer's helpful comments to improve our manuscript and for their time reviewing our submission. Thank you very much!

Reviewer 3 Report

Authors have addressed most of the comments. However, the Fig.4 looks of poor quality when the manuscript is printed. The Fig. 3c is confusing. It seems that the red circles visually interferes with the image.  Also, most of the references are not recent. 

Author Response

We appreciate the reviewer's helpful comments to improve our manuscript and for their time reviewing our submission. Figure 1c has been edited to show thinner, dashed red lines and a description was added to the figure legend as follows:

Red dashed lines indicate the outline of the HBVSMC/collagen gels which were used to calculate the area.

We apologize for the misunderstanding regarding incorporation of references. The newer references are now incorporated in the reference list

Thank you very much!