Review Submission

16/01/12 12:10

Bertrand Gondouin (Author) Queue SummaryReviewer Area

Comments for the Author

Return 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 DignatGeorge, 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 Biology Corresponding 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 <100 M (25 !g/ml) (Figure 1C). However, 25 !g/ml of IS does not appear to have a significant effect on TF expression in vitro (Figure 2A). Furthermore, all of the other in vitro experiments appear to have been carried out with the highest concentrations of IS (250 !g/ml) and IAA (9 !g/ml). These are much higher than were found in vivo. In addition, since uremic solutes have been previously shown to be detrimental to endothelial cells, did the addition of
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previously shown to be detrimental to endothelial cells, did the addition of 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 Tissue Factor (sTF) is linked to increased mortality in patients with coronary
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Tissue Factor (sTF) is linked to increased mortality in patients with 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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