Reviewer ’s report  
 
Title: Anti-fibrogenic effect of PPAR -γ agonists in human intestinal  myofibroblasts  
 
Version : 0 Date: 28 Dec 2016  
 
Reviewer: Eric Smith  
 
Reviewer's report:  
 
Intestinal fibrosis is a common complication of inflammatory bowel disease that may 
significantly reduce quality of life and require invasive therapeutic intervention. Chronic 
inflammation is known to activate fibro blasts, inducing a contractile myofibroblast phenotype 
and increasing the deposition of extracellular matrix (ECM). PPAR -gamma agonists may modify 
fibrosis.  
 
 
 
The aim of this study was to determine if the PPAR -gamma synthetic agonists troglitazone 
(TRG) and  rosiglitazone (RSG) could inhibit the activation of primary human intestinal 
fibroblasts (HIFs) and elucidate the mechanisms involved.  
 
 
 
The authors demonstrated that TRG and RSG inhibited the TGF -beta1 -induced expression of the 
activated myofibroblast ma rker alpha -smooth muscle actin (alpha -SMA) and the ECM proteins 
collagen 1A1 and fibronectin. TRG and RSG also inhibited the TGF -beta1 -induced 
phosphorylation of Smad2 and Akt. Pretreatment of the HIFs with the highly specific and 
irreversible PPAR -gamma a ntagonist GW9662 failed to block the observed inhibition, 
suggesting that the observed inhibitory effects of TRG and RSG were independent of PPAR -
gamma activation. Although PPAR -gamma synthetic agonists have previously been shown to 
inhibit TGF -beta1 -induc ed effects independent of PPAR -gamma activation, to the best of my 
knowledge this is the first study to demonstrate this effect in human intestinal fibroblasts.   
 
 
 
The authors state that "we showed that PPAR -gamma agonists suppress TGF -beta1 -induced 
synthe sis of ECM and alpha -SMA in HIFs by blocking Akt phosphorylation".  However, in 
Figure 5B they demonstrated that pretreatment with 50uM of the PI3K inhibitor LY294002, a 
dose which was sufficient to inhibit the TGF -beta1 -induced expression of collagen 1A, 
fibronectin and alpha -SMA, did not block the TGF -beta1 -induced phosphorylation of Akt.  
Based on the available data, p -Akt alone does not appear to be sufficient to induce the expression 
of alpha -SMA, collagen 1A1 and fibronectin. Therefore, there is insuf ficient evidence to suggest 
that the PPAR -gamma agonists suppresses TGF -beta1 -induced synthesis of ECM and alpha -
SMA in HIFs by blocking Akt phosphorylation. This needs to be addressed throughout the 
manuscript.  
The authors proposed a model (figure 7) in which PPAR -gamma agonists suppress activation 
Smad2 and Akt. However, it is unclear if all PPAR -gamma agonists act in this manner, or if this 
finding is restricted to TRG and RSG. The figure should be modified to reflect this.  
 
 
 
The concluding statement that "Our results strongly suggest that the synthetic PPAR -gamma 
agonists, TRG and RSG, could be promising anti -fibrotic agents to prevent or treat intestinal 
fibrosis" is unsubstantiated by the data presented and should be modifie d. 
 
 
 
 
Corrections:  
 
Page 18, line 36 "revered" should be "reversed".  
 
 
 
 
Are the methods appropriate and well described?  
If not, please  specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspe cts require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete  a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for an y patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competin g interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachm ents I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommon s.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Anti-fibrogenic effect of PPAR -γ agonists in human intestinal  myofibroblasts  
 
Version : 0 Date: 28 Dec 2016  
 
Reviewer: Eric Smith  
 
Reviewer's report:  
 
Intestinal fibrosis is a common complication of inflammatory bowel disease that may 
significantly reduce quality of life and require invasive therapeutic intervention. Chronic 
inflammation is known to activate fibro blasts, inducing a contractile myofibroblast phenotype 
and increasing the deposition of extracellular matrix (ECM). PPAR -gamma agonists may modify 
fibrosis.  
 
 
 
The aim of this study was to determine if the PPAR -gamma synthetic agonists troglitazone 
(TRG) and  rosiglitazone (RSG) could inhibit the activation of primary human intestinal 
fibroblasts (HIFs) and elucidate the mechanisms involved.  
 
 
 
The authors demonstrated that TRG and RSG inhibited the TGF -beta1 -induced expression of the 
activated myofibroblast ma rker alpha -smooth muscle actin (alpha -SMA) and the ECM proteins 
collagen 1A1 and fibronectin. TRG and RSG also inhibited the TGF -beta1 -induced 
phosphorylation of Smad2 and Akt. Pretreatment of the HIFs with the highly specific and 
irreversible PPAR -gamma a ntagonist GW9662 failed to block the observed inhibition, 
suggesting that the observed inhibitory effects of TRG and RSG were independent of PPAR -
gamma activation. Although PPAR -gamma synthetic agonists have previously been shown to 
inhibit TGF -beta1 -induc ed effects independent of PPAR -gamma activation, to the best of my 
knowledge this is the first study to demonstrate this effect in human intestinal fibroblasts.   
 
 
 
The authors state that "we showed that PPAR -gamma agonists suppress TGF -beta1 -induced 
synthe sis of ECM and alpha -SMA in HIFs by blocking Akt phosphorylation".  However, in 
Figure 5B they demonstrated that pretreatment with 50uM of the PI3K inhibitor LY294002, a 
dose which was sufficient to inhibit the TGF -beta1 -induced expression of collagen 1A, 
fibronectin and alpha -SMA, did not block the TGF -beta1 -induced phosphorylation of Akt.  
Based on the available data, p -Akt alone does not appear to be sufficient to induce the expression 
of alpha -SMA, collagen 1A1 and fibronectin. Therefore, there is insuf ficient evidence to suggest 
that the PPAR -gamma agonists suppresses TGF -beta1 -induced synthesis of ECM and alpha -
SMA in HIFs by blocking Akt phosphorylation. This needs to be addressed throughout the 
manuscript.  
The authors proposed a model (figure 7) in which PPAR -gamma agonists suppress activation 
Smad2 and Akt. However, it is unclear if all PPAR -gamma agonists act in this manner, or if this 
finding is restricted to TRG and RSG. The figure should be modified to reflect this.  
 
 
 
The concluding statement that "Our results strongly suggest that the synthetic PPAR -gamma 
agonists, TRG and RSG, could be promising anti -fibrotic agents to prevent or treat intestinal 
fibrosis" is unsubstantiated by the data presented and should be modifie d. 
 
 
 
 
Corrections:  
 
Page 18, line 36 "revered" should be "reversed".  
 
 
 
 
Are the methods appropriate and well described?  
If not, please  specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspe cts require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete  a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for an y patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competin g interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachm ents I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommon s.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Anti-fibrogenic effect of PPAR -γ agonists in human intestinal  myofibroblasts  
 
Version : 0 Date: 28 Dec 2016  
 
Reviewer: Eric Smith  
 
Reviewer's report:  
 
Intestinal fibrosis is a common complication of inflammatory bowel disease that may 
significantly reduce quality of life and require invasive therapeutic intervention. Chronic 
inflammation is known to activate fibro blasts, inducing a contractile myofibroblast phenotype 
and increasing the deposition of extracellular matrix (ECM). PPAR -gamma agonists may modify 
fibrosis.  
 
 
 
The aim of this study was to determine if the PPAR -gamma synthetic agonists troglitazone 
(TRG) and  rosiglitazone (RSG) could inhibit the activation of primary human intestinal 
fibroblasts (HIFs) and elucidate the mechanisms involved.  
 
 
 
The authors demonstrated that TRG and RSG inhibited the TGF -beta1 -induced expression of the 
activated myofibroblast ma rker alpha -smooth muscle actin (alpha -SMA) and the ECM proteins 
collagen 1A1 and fibronectin. TRG and RSG also inhibited the TGF -beta1 -induced 
phosphorylation of Smad2 and Akt. Pretreatment of the HIFs with the highly specific and 
irreversible PPAR -gamma a ntagonist GW9662 failed to block the observed inhibition, 
suggesting that the observed inhibitory effects of TRG and RSG were independent of PPAR -
gamma activation. Although PPAR -gamma synthetic agonists have previously been shown to 
inhibit TGF -beta1 -induc ed effects independent of PPAR -gamma activation, to the best of my 
knowledge this is the first study to demonstrate this effect in human intestinal fibroblasts.   
 
 
 
The authors state that "we showed that PPAR -gamma agonists suppress TGF -beta1 -induced 
synthe sis of ECM and alpha -SMA in HIFs by blocking Akt phosphorylation".  However, in 
Figure 5B they demonstrated that pretreatment with 50uM of the PI3K inhibitor LY294002, a 
dose which was sufficient to inhibit the TGF -beta1 -induced expression of collagen 1A, 
fibronectin and alpha -SMA, did not block the TGF -beta1 -induced phosphorylation of Akt.  
Based on the available data, p -Akt alone does not appear to be sufficient to induce the expression 
of alpha -SMA, collagen 1A1 and fibronectin. Therefore, there is insuf ficient evidence to suggest 
that the PPAR -gamma agonists suppresses TGF -beta1 -induced synthesis of ECM and alpha -
SMA in HIFs by blocking Akt phosphorylation. This needs to be addressed throughout the 
manuscript.  
The authors proposed a model (figure 7) in which PPAR -gamma agonists suppress activation 
Smad2 and Akt. However, it is unclear if all PPAR -gamma agonists act in this manner, or if this 
finding is restricted to TRG and RSG. The figure should be modified to reflect this.  
 
 
 
The concluding statement that "Our results strongly suggest that the synthetic PPAR -gamma 
agonists, TRG and RSG, could be promising anti -fibrotic agents to prevent or treat intestinal 
fibrosis" is unsubstantiated by the data presented and should be modifie d. 
 
 
 
 
Corrections:  
 
Page 18, line 36 "revered" should be "reversed".  
 
 
 
 
Are the methods appropriate and well described?  
If not, please  specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspe cts require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
Declaration of competing interests  
Please complete  a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for an y patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competin g interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachm ents I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommon s.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant