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Bertrand Gondouin (Author)Queue SummaryReviewer Area
Comments for the AuthorReturn to Queue
BLOOD/2011/394726
Role of indolic uremic solutes on tissue factor production via aryl hydrocarbon receptor pathway
Bertrand Gondouin, Claire Cerini, Laetitia Dou, Ariane Duval-Sabatier, Anneleen Pletinck, Raymond Calaf, Noemie Jourde,
Stephane Poitevin, Laurence Camoin-Jau, Laurent Arnaud, Raymond Vanholder, Philippe Brunet, Francoise Dignat-
George, and Stephane Burtey
Decision: Reject; Decision Date: 31 Dec 2011
Date Received: 9 Dec 2011
Editor: David Lillicrap
Article Type: Regular Article
Secondary Scientific Category: Thrombosis and Hemostasis
Primary Scientific Category: Vascular BiologyCorresponding Author: Stephane Burtey
Keywords: COAGULATION, Biochemistry and structural biology of hemostatic proteins, Coagulation co-factors;
VASCULAR BIOLOGY; VASCULAR BIOLOGY, Endothelial cells; Indole acetic acid; Indoxyl sulfate; Tissue factor; aryl
hydrocarbon receptor; chronic kidney disease
Supplemental Files: 0
Reviewer 1 Comments for the Author
Reviewer 2 Comments for the Author
Reviewer 3 Comments for the Author
Reviewer 1 Comments for the Author...
The manuscript describes a correlation between plasma TF levels and levels of
the uremic solutes IS and IAA in patients with chronic kidney disease and shows
the upregulation of cell surface TF in endothelial cells and PBMC in response
to IS and IAA. The authors also describe a new signalling mechanism leading to
TF expression involving activation of the aryl hydrocarbon receptor. However,there are some points that need addressing:
Major points:
1. The maximum concentrations of IS (1 mM or 250 !g/ml) and IAA (50 M or 9
!g/ml) used for the in vitro experiments are above the physiological
concentrations found in the serum of CKD patients as shown in Figure 1. In
fact, the highest concentration of IS found in vivo was
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these concentrations of uremic solutes affect cell viability?
2. Figure 6G shows TF activity of cells transfected with siRNA and treated with
IAA. Was TF mRNA and protein expression also reduced in these cells following
treatment with IAA? Figures 6B and 6C show the levels of AHR expression 48 h
after transfection of cells with the control and AHR siRNA. However, in the
methods section it is stated that the effects on TF expression were carried out
72 h after transfection. AHR mRNA and protein levels should therefore also be
shown at 72 h post-transfection with siRNA. Figure 7 shows only TF mRNA and TF
activity. Was TF protein expression also reduced following treatment of cells
with TCDD?
3. Figure 5 shows that the NFkB inhibitor (10 !M) wedelolactone only
partially inhibited TF expression in response to IS and IAA in HUVEC. Was this
concentration of wedelolactone shown to completely inhibit NFkB in these cells?
A dose-response curve is needed to prove that complete inhibition of NFkB only
partially blocks TF upregulation by IS and IAA.
4. In the introduction it is stated that uremic solutes have been shown to
induce the release of microparticles from endothelial cells. The effect of
uremic solutes on the release of TF-positive microparticles from endothelial
cells and PBMC would strengthen this study, and possibly explain increased
plasma TF levels in CKD patients.
5. The authors identify the AHR signalling pathway as a modulator of TF
expression in response to IS and IAA. However, since there are no known AHR
response elements in the promoter of the TF gene, how do the authors propose
that AHR activation upregulates the expression of TF?
6. Does sTF refer to full-length TF associated with microparticles, or
alternatively spliced TF in the plasma? Did sTF in plasma
have procoagulant activity?
Minor points:
1. Error in the x-axis of Figure 2A and in the y-axis of Figure 2F.
2. In the abstract the authors have stated that TF production is increased in
endothelial and PBMC via AHR activation. However, they have not shown the
involvement of AHR signalling in PBMC.
Reviewer 2 Comments for the Author...
This study analyzes the regulation of tissue factor (TF) by indole uremic solutes.
Major comments
1/ A commercial ELISA is used to measure soluble TF levels in plasma.
Significant concerns have been raised about the specificity of this ELISA.
Therefore, other measures of circulating TF should be performed to confirm the
observed changes.
2/ There is considerable variation in the basal levels of TF protein (25-125
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pg/ml) and TF activity (10-50 ng/ml) and fold changes in the different
experiments. This is important because some of the changes are quite modest and
would be lost if there is a high basal expression. The SD in some experiments is
also very large (i.e. Figure 2E).
3/ The in vitro studies have focused on ECs but PBMCs generally express higher
levels of TF. More studies should be performed with PBMCs.
4/ The western blot shown in Figure 2C is not convincing because TF is
glycosylated and runs as a smear rather than a distinct band. How many
experiments were performed?
5/ More than one AhR siRNA should be used. Where is the control without siRNA?
6/ Do IS and IAA activate NF-KB?
7/ It is unclear why the AHR inhibitor abolishes TF induction whereas the NF-KB
only give partial inhibition. Why does the AHR inhibitor reduce basal TF
expression? Has toxicity been examined?
8/ Has LPS contamination been excluded?
9/ Has an AHR site been identified in the TF promoter?
10/ Please remove for the first time from the manuscript.
Minor comments
1/ Y axis of Figure 2F.
2/ Why is the control in Figure 4A not 1?
3/ Wededlolactone is not a typical NF-KB inhibitor. Provide a reference. Other
inhibitors should be used.
4/ Y axis of Figure 6 B should be decrease.
5/ Error bars need to be added to the controls in Figure 7A.
6/ When are the samples collected relative to dialysis?
7/ HUVECs are prepared not extracted.
Reviewer 3 Comments for the Author...
Cardiovascular mortality is substantially increased in patients with chronic
kidney disease (CKD), however, the underlying mechanisms are still poorly
understood. Here, Gondouin and colleagues investigated in 73 hemodialysis (HD)
patients and 50 non-dialized CKD patients whether indolic uremic solutes induce
Tissue Factor (TF) production and thereby shifting the coagulation cascade in
CKD patients towards a pro-trombotic state. Since elevated levels of soluble
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Tissue Factor sTF is in e to increase morta ity in patients wit coronary
heart disease, an elevated production of sTF under uremic conditions may also
add to the increased cardiovascular mortality in CKD patients.
The authors convincingly demonstrated that sTF is elevated in HD and undialyzed
CKD patients compared to controls and that sTF is correlated with indoxyl
sulfate (IS) in CKD patients and indole-3-acetic acid (IAA) in HD patients. Both
indolic uremic solutes can induce TF expression and subsequent procoagulant
activity in endotheial vells under in vitro conditions. The expression of TF
through both indolic uremic solutes can be reduced but not completely inhibited
by wedelolactone, an inhibitor of the NF-kB signalling pathway. Microarray
experiments using HUVEC incubated with IS revealed an up-regulation of genes
regulated by the transcription factor aryl hydrocarbon receptor (AHR), a major
mediator of organism response to xenobiotics. Here the authors demonstrate for
the first time that indolic uremic solutes modulate TF production via the AHR
pathway.
Specific comments:
1) What genes are up- regulated after incubation of HUVECS with IAA for 4 hours?
How many of these genes match with the ones seen in the IS experiments.
2) The authors have nicely shown the effect of AHR on IS mediated TF production.But what role plays AHR on the IAA mediated TF production. The authors should
also measure the effect of AHR silencing on TF production.
3) Since PBMNC are easily available form HD and CKD patients, the authors should
repeat their AHR silencing experiments on TF expression and activity induced by
IS and IAA in PBMNCs.
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