EMBO reports - Peer Review Process File - EMBOR-2011-35207

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Manuscript EMBOR-2011-35207 Tie1 Deficiency Induces Endothelial-Mesenchymal-Transition Julie Garcia, Maria Jose Sandi, Pierre Cordelier, Bernard Binetruy, Jacques Pouyssegur, Juan Lucio Iovanna and Roselyne Tournaire Corresponding author: Roselyne Tournaire, INSERM U624 Review timeline: Submission date: 08 July 2011 Editorial Decision: 10 August 2011 Revision received: 07 November 2011 Editorial Decision: 02 December 2011 Revision received: 13 February 2012 Accepted: 14 February 2012 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision 10 August 2011

Thank you for the submission of your manuscript to our offices. I would like to apologize for the delay in getting back to you, but we have just now received the full set of reports from the referees, which I copy below. As two of the referees consider the manuscript highly interesting and the third referee express concerns that are, in principle addressable, I would like to ask you to revise it according to the referees' comments. One of the major concerns raised by both referee #1 and #3 regards the interpretation of the data in pancreatic cancer tissues. They both agree in that further experimental work would be necessary, particularly concerning the use of more markers to fully assess the identity of the cells observed. Also related to this experiments, referee #1 considers that further description of the tumors used is needed. Besides the above referred issue and general concerns about the technical quality of the experiments described, referee #2 also remarks that the assessment of the change in phenotype of endothelial cells is incomplete and more markers need to be provided to fully demonstrate this point. Given these evaluations and the potential interest of your study, I would like to give you the opportunity to revise your manuscript, with the understanding that the referee concerns must be fully addressed and their suggestions (as detailed above and in their reports) taken on board. Acceptance of the manuscript will depend on a positive outcome of a second round of review and I should also remind you that it is EMBO reports policy to allow a single round of revision only and that,

EMBO reports - Peer Review Process File - EMBOR-2011-35207

© European Molecular Biology Organization

therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I look forward to seeing a revised version of your manuscript when it is ready. Yours sincerely, Editor EMBO reports REFEREE REPORTS: Referee #1: In this ms. Garcia et al. analyze the mechanisms involved in the endothelial-mesenchymal transition using a variety of endothelial cell cultures and assess the role of Tie1 in the maintenance of the endothelial phenotype. They show, using a variety of methods, that upon Tie1 down-regulation Snai2 is increased and mediates - in part - an endothelial-mesenchymal transition. Using siRNA strategies and chemical inhibitors they show that Erk5 plays an important role in this process. They then go on to show that cells displaying at the same time markers of endothelial cells and myofibroblasts are detected in pancreatic tumor stroma and they suggest that this results from endothelial-mesenchymal transitions. They also invoke a putative role of a lncRNA recently reported to regulate Tie1. The work is novel and interesting and provides new hypotheses regarding the role of endothelial-mesenchymal transitions in tumor biology. The paper is well written and clear. However, there are a number of issues that the authors should address, as specified below. Specific comments 1. The authors should provide evidence of the specificity of the antibodies against Erk5 and of the Erk5 knockdown by assessing, at least, Erk1/2 expression in the western blots in which they assess Erk5 down-regulation (Figure 4). 2. The extension of the study to pancreas cancer tissues is highly interesting but data interpretation is flawed. First, since they find that only a proportion of CD31+ cells express fibroblast markers (and one would expect this to be a transient phenotype), the same types of quantitative analyses should be performed on endothelial cells from normal tissues where an endothelial-mesenchymal transition is not expected to tak place. Second, if additional markers were used in the co-expression studies the data would be more convincing because S100A4 is not totally fibroblast-specific (see Human Protein Atlas data page). Third, even if additional markers are used, an endothelial-mesenchymal transition cannot be formally proven and this has to be said. There is data interpretation in these studies, of course this can be speculated but it needs to be clearly stated in the text. Finally, the lncRNA analysis is rather incomplete and it does not contribute to clarify the mechanism involved in the putative endothelial-mesenchymal transition occurring in tumor tissues. 3. The information on the tumor tissues used should be provided. In addition, more information on the cellular composition of the tissues used for analyses should be included: did the authors use primary tumor from patients with metastases? were the tissues comparable to those from those patients without metastases? how is expression in metastases vs. primary tumors? The comparison with normal pancreas should also be clarified. Minor comments 1. In the main text, the authors should provide a rationale for focusing on Erk5 prior to reporting the experiments. Because there are good evidences supporting a role for this MAP kinase in endothelial differentiation, this should help to substantiate their studies. 2. The description in the main text of the adherence experiments should be briefly expanded and improved, there should be no need to go to the Suppl. material. 3. S100A4 is the same gene/protein as FSP1; therefore, a uniform nomenclature should be used throughout the paper and the point should be made (if necessary) that both designations correspond

EMBO reports - Peer Review Process File - EMBOR-2011-35207

© European Molecular Biology Organization

to the same molecular species. Referee #2: The manuscript by Tournaire and colleagues addresses the interesting question of endothelial to mesenchymal transition, using in vitro studies on human endothelial cells and some observations in human pancreatic cancer. The authors report the interesting finding that induced deficiency in Tie1 expression by siRNA leads to loss of VE-cadherin expression and upregulation of mesenchymal markers. Tie1 deficiency increased cell migration and responsiveness to PDGFB. The effects were reverted by simultaneous knockdown of Slug. Studying the signalling pathways involved in Slug induction upon Tie1 deficiency, the authors show that Erk1/2 and especially Erk5 activity is required for the effects. Finally the authors show stainings for CD31, Tie1, Tie2, aSMA, and S100A4 on human pancreatic cancer samples to investigate evidence for endoMT and report on increased expression of a previously described antisense RNA for Tie1 in metastatic tumours. Overall, this is a timely study into the molecular regulation of the interesting phenomenon of endoMT, providing first evidence for the involvement of Tie1 regulation. The paper is concise and clearly written and the data well illustrated. The conclusions on endoMT in vivo are based on indirect evidence, but without moving into experimental in vivo models, it will be difficult to improve on that. One would like to see a mouse tumour model and lineage tracing using Tie1 loss of function to study this process in a more rigorous in vivo setting. However, in its current form, the manuscript is strongest on the in vitro investigation of the molecular link. This referee believes the data will be of interest to the field. Specific comments Figure 1: it would be good to mention or show also the results obtained for the vascular and mesenchymal markers after Tie-2 knockdown for direct comparison. Further, do BAE cells in addition to the morphological changes also show the same marker profile changes? Figure 2: Given that figure 1 uses the morphology of cells as readout, it seems important to show that simultaneous KD of Slug reverts the morphological changes as well. Figure 3: Is this random migration or directional migration? The text doesn't state this clearly. Does the induction of sensitivity towards PDGFB correlate with up-regulation of the relevant receptor? Figure 5: This figure apparently contains stainings on consecutive sections of the same tumour area in A and B. The indicated cell in top panel in A is the same cell as in the second row of B. Only the orientation is different as the image is rotated 180 degrees. Different stainings of the same cell should be indicated in the legend. In general using the very same cell in two panels without indicating gives the impression that this could be a very rare event. Referee #3: Garcia et al. explore the effect of Tie1 KD in endothelial cells. They find that loss of Tie-1 not Tie-2 induced Endo-MT. They also found that loss of Tie-1 activates ERK1/2, ERK5, and Akt and leads to induction of Slug promoter activity. In vivo, the authors find that Endo-MT contributes to human pancreatic tumor formation. In general, the data are poorly presented and figures are poorly organized. There are major problems with both in vitro and in vivo data and the data, as presented, do not support the conclusions. Major 1. Fig 1: gene expression changes after Tie1 knockdown. A potential contamination with non-EC needs to be ruled out. Furthermore, all changes in gene expression need to be confirmed by Western blotting. Does Tie1 KD induce EC apoptosis? 2. Supplementary Figure S1 only looks at cell morphology, cell migration, ERK1/2 activity. How

EMBO reports - Peer Review Process File - EMBOR-2011-35207

© European Molecular Biology Organization

about smooth muscle markers, mesenchymal markers, ERK5, Akt activity? 3. 4. The effect of Slug KG in Tie1 KD cells on gene expression is totally unconvincing (with exception of collagen). Once again, the data is presented very poorly. 5. Since TGFbeta signaling is known to play a major role in End0-MT, this signaling cascade needs evaluated after Tie1 KD. 6. Fig 3: the effect of Tie1 KD on cell migration is measured in the PDGF stimulation assay. As normal EC express very little, if any, PDGF-R, the differences in migration may be simply due to PDGF-R expression levels that have not been assessed. The migration assay is not described but looks like a Boyden chamber assay. Instead, the authors should examine unstimulated migration and migration in response to VEGF and FGF (after first checking receptor levels before and after Tie 1KD) in an in vitro wounding assay 7. Fig 3 adhesion assay: why choose Fn as the matrix for this assay? Several different matrices need to be assayed and expression of corresponding integrins determined. 8. Fig 4: what time point is shown? In general, a time course is required for these types of studies. Furthermore, Studies using high dose (20 microM) chemical inhibitors are really not acceptable without confirmation using dominant-negative constructs or something similar. 9. Fig 5: The authors need to show that Tie1 knockdown in EC inducea Endo-MT in vivo. This is absolutely critical. -Figure 5A and Figure 5C do not provide any useful information and should be removed. -Li et al (Blood 2010) showed that human cells have 3 Tie-1 RNA transcripts. Do all 3 transcripts increase in human pancreatic tumor + metastasis samples? Are those RNA samples from whole tumors or isolated primary EC from the metastasis tumors? -The authors measure Tie-1 AS lncRNA expression by qPCR. What is the specificity of this assay? -The authors state that Endo-MT is characterized by loss of CD31 in the introduction and show that Tie-1 knockdown results in a loss of CD31 in EC undergo Endo-MT. On page 8, the authors state that EC undergo Endo-MT have both CD31 and FSP1/SMA markers. What gives? -the authors use the term "complete phenotype switch" to characterize the CD31-/SMA+ or CD31+/S100A4 cell population as cells undergo Endo-MT. Without EC fate mapping, how do the authors know the cells are EC, especially since Tie-1 and Tie-2 are not specific EC markers? Fate mapping is absolutely required for End0-MT studies in vivo. Minor 1. All figures are unnumbered which makes it rather hard on the reader. 2. Fig 1C is very poorly put together 3. Fig 2A presentation is very poor. It looks like there is a massive increase in Snail expression until one notices the scale. 4. The authors use HMVECs cells: what is the purity and passage number of those cells? What is the source, purity, and passage number of the BAE cells? 5. Figure 5B and Figure 5C descriptions in text do not match the figure legend. 1st Revision - authors' response 07 November 2011

Referee #1:

In this ms. Garcia et al. analyze the mechanisms involved in the

endothelial-mesenchymal transition using a variety of endothelial cell

cultures and assess the role of Tie1 in the maintenance of the endothelial

phenotype. They show, using a variety of methods, that upon Tie1 down-

regulation Snai2 is increased and mediates - in part - an endothelial-

mesenchymal transition. Using siRNA strategies and chemical inhibitors they

show that Erk5 plays an important role in this process. They then go on to

show that cells displaying at the same time markers of endothelial cells

and myofibroblasts are detected in pancreatic tumor stroma and they suggest

that this results from endothelial-mesenchymal transitions. They also

invoke a putative role of a lncRNA recently reported to regulate Tie1. The

work is novel and interesting and provides new hypotheses regarding the

role of endothelial-mesenchymal transitions in tumor biology. The paper is

well written and clear. However, there are a nu

mber

of issues that the authors should address, as specified below.

Specific comments

1. The authors should provide evidence of the specificity of the antibodies

against Erk5 and of the Erk5 knockdown by assessing, at least, Erk1/2

expression in the western blots in which they assess Erk5 down-regulation

(Figure 4).

We agree with the comment of this reviewer about the specificity of ERK5 antibodies. In fact, these antibodies are specific because they recognize only the ERK5 band at 115 Kd and no ERK1/2 band at 42/44 Kd. This is shown in Additional data for the referees at the end of our responses (Figure 1). Concerning the second question, we have now added a blot in Fig 3D showing that ERK5 deficiency does not modified Erk1/2 expression.

2. The extension of the study to pancreas cancer tissues is highly

interesting but data interpretation is flawed. First, since they find that

only a proportion of CD31+ cells express fibroblast markers (and one would

expect this to be a transient phenotype), the same types of quantitative

analyses should be performed on endothelial cells from normal tissues where

an endothelial-mesenchymal transition is not expected to tak place.

This is a very interesting comment. However, it is technically and ethically challenging to obtain human normal pancreas tissue. One way to circumvent this problem is to look an area without histological evidence of the presence of tumoral lesions the most distant from the tumor. We have

performed these experiments and the results show that there is no co-staining SMA+/CD31+ in the same cell. These results have been added in Fig 4B.

Second, if additional markers were used in the co-expression studies the

data would be more convincing because S100A4 is not totally fibroblast-

specific (see Human Protein Atlas data page).

We thank the referee for this very useful suggestion. We have now analyzed more markers to assess the identity of the observed cells. We have selected N-cadherin, FAP (Fibroblast activation protein)

and SM22. All these markers have been evaluated in co-staining experiments with CD31. Experiments revealed the coexpression of CD31 with several mesenchymal markers in a sub-

population of cells: CD31+/SMA+, CD31+/S100A4 +, CD31+/N-Cad+, CD31+/FAP+, CD31+/SM22+. This coexpression in cells was detectable in association with Tie2 but never with Tie1. These results have been added in Fig 4 and Fig 5.

Third, even if additional markers are used, an endothelial-mesenchymal

transition cannot be formally proven and this has to be said. There is data

interpretation in these studies, of course this can be speculated but it

needs to be clearly stated in the text.

We agree with the reviewer and have now changed the text. We describe a cell population which expresses both endothelial and mesenchymal markers and we speculate that this population could derive from an endothelial-mesenchymal transition. Furthermore, Zeisberg et al. showed that, in endothelial cell–specific LacZ reporter mice, tumors contain fibroblasts that are also LacZ, thereby demonstrating their endothelial origin (Zeisberg et al., 2007). We have added this sentence in the text. Finally, the lncRNA analysis is rather incomplete and it does not

contribute to clarify the mechanism involved in the putative endothelial-

mesenchymal transition occurring in tumor tissues.

In light of reviewers 1 and 3 comments concerning the lncRNA analysis in pancreatic tumors, we decided to remove these experiments because we agree that these results are inconclusive and do not prove a mechanism for the inhibition of Tie1 in pancreatic tumors.

3. The information on the tumor tissues used should be provided. In

addition, more information on the cellular composition of the tissues used

for analyses should be included: did the authors use primary tumor from

patients with metastases? were the tissues comparable to those from those

patients without metastases? how is expression in metastases vs. primary

tumors? The comparison with normal pancreas should also be clarified.

We thank the referee for this very useful suggestion. In fact, we recognize that we described very poorly the tumors. We have now included a table in Supplementary Information section (Table S1) that provides details about the tumors used for immuno fluorescence staining. A TNM classification was used (UICC 2009). Minor comments

1. In the main text, the authors should provide a rationale for focusing on

Erk5 prior to reporting the experiments. Because there are good evidences

supporting a role for this MAP kinase in endothelial differentiation, this

should help to substantiate their studies.

This is a very useful suggestion. We have now added two sentences and three references to explain why we have analyzed the effects of Tie1 deficiency on ERK5 phosphorylation: “Studies have shown that the ERK5 pathway is critical for normal cardiovascular development and vascular integrity (Regan et al., 2002; Nishimoto and Nishida, 2006) and that it can control Slug expression (Arnoux et al., 2007).

2. The description in the main text of the adherence experiments should be

briefly expanded and improved, there should be no need to go to the Suppl.

material.

Because the adhesion test is already described, we believe that the referee is speaking about the migration test. In fact we recognize that this migration test was very little described. We added a paragraph in the Methods section describing the migration test used and these words “in a modified Boyden chamber assay” in the legend to Fig 2D. 3. S100A4 is the same gene/protein as FSP1; therefore, a uniform

nomenclature should be used throughout the paper and the point should be

made (if necessary) that both designations correspond to the same molecular

species.

We agree with you. We have corrected our article and we use S100A4 stating that S100A4 and FSP1 correspond to the same molecular species.

Referee #2:

The manuscript by Tournaire and colleagues addresses the interesting

question of endothelial to mesenchymal transition, using in vitro studies

on human endothelial cells and some observations in human pancreatic

cancer. The authors report the interesting finding that induced deficiency

in Tie1 expression by siRNA leads to loss of VE-cadherin expression and

upregulation of mesenchymal markers. Tie1 deficiency increased cell

migration and responsiveness to PDGFB. The effects were reverted by

simultaneous knockdown of Slug. Studying the signalling pathways involved

in Slug induction upon Tie1 deficiency, the authors show that Erk1/2 and

especially Erk5 activity is required for the effects. Finally the authors

show stainings for CD31, Tie1, Tie2, aSMA, and S100A4 on human pancreatic

cancer samples to investigate evidence for endoMT and report on increased

expression of a previously described antisense RNA for Tie1 in metastatic

tumours.

Overall, this is a timely study into the molecular regulation of the

interesting phenomenon of endoMT, providing first evidence for the

involvement of Tie1 regulation. The paper is concise and clearly written

and the data well illustrated.

The conclusions on endoMT in vivo are based on indirect evidence, but

without moving into experimental in vivo models, it will be difficult to

improve on that.

One would like to see a mouse tumour model and lineage tracing using Tie1

loss of function to study this process in a more rigorous in vivo setting.

However, in its current form, the manuscript is strongest on the in vitro

investigation of the molecular link. This referee believes the data will be

of interest to the field.

Specific comments

Figure 1: it would be good to mention or show also the results obtained for

the vascular and mesenchymal markers after Tie-2 knockdown for direct

comparison. Further, do BAE cells in addition to the morphological changes

also show the same marker profile changes?

We performed these experiments and results show that Tie2 deficiency doesn’t induce modification

on the mRNA and protein expression of endothelial (CD31, VE-Cad, CD34, FVIII) and mesenchymal (

SMA, S100A4, Col1a1, SM22 N-cadherin) markers. These results have been added in Fig 1D. Concerning BAE cells, Tie1 deficiency strongly decreases the protein expression of the vascular endothelial markers(CD31, VE-Cad, CD34, FVIII), and increases expression of the mesenchymal

markers (SMA, S100A4, Col1a1, SM22N-cadherin). These results have been added in Supplementary Fig S1B. We also show that Tie deficiency in BAE cells induces Akt and ERK5 phosphorylation (Supplementary Fig S1E). Figure 2: Given that figure 1 uses the morphology of cells as readout, it

seems important to show that simultaneous KD of Slug reverts the

morphological changes as well.

We thank the referee. We have included a picture in Fig 2B showing that Slug deficiency reverts the morphological changes induced by Tie1 down regulation.

Figure 3: Is this random migration or directional migration? The text

doesn't state this clearly.

In fact, we recognize that this migration test was very little described. We added a paragraph in the Methods section describing the migration test used and these words “in a modified Boyden chamber

assay” in the legend to Fig 2D. We chose to use a directional test because PDGF is a chemotactic factor and PDGF-directional test is routinely used to assess migration (Ball et al., 2007; Veevers-Lowe et al., 2011). Does the induction of sensitivity towards PDGFB correlate with up-

regulation of the relevant receptor?

This is a very useful suggestion. Indeed the increased migration induced by Tie1 deficiency can be explained by an increase in PDGF receptors: we have now performed these experiments and we

show that Tie1 deficiency increases protein expression of the PDGFR receptor . These results are added in Fig 2D.

Figure 5: This figure apparently contains stainings on consecutive sections

of the same tumour area in A and B. The indicated cell in top panel in A is

the same cell as in the second row of B. Only the orientation is different

as the image is rotated 180 degrees. Different stainings of the same cell

should be indicated in the legend. In general using the very same cell in

two panels without indicating gives the impression that this could be a

very rare event.

We thank and agree with the referee. We have removed this picture. As a result of the rarity of

frozen pancreatic tissue samples, we use serial sections as it was the case for the slides you’re talking

about; that is why the same cell was presented on two different slides. We looked at other cells and

we show now the co-staining in different cells (Fig 4A and 5).

Referee #3:

Garcia et al. explore the effect of Tie1 KD in endothelial cells. They find

that loss of Tie-1 not Tie-2 induced Endo-MT. They also found that loss of

Tie-1 activates ERK1/2, ERK5, and Akt and leads to induction of Slug

promoter activity. In vivo, the authors find that Endo-MT contributes to

human pancreatic tumor formation.

In general, the data are poorly presented and figures are poorly organized.

There are major problems with both in vitro and in vivo data and the data,

as presented, do not support the conclusions.

Major

1. Fig 1: gene expression changes after Tie1 knockdown. A potential

contamination with non-EC needs to be ruled out.

We used HMVECs which is an established cell line isolated after immortalization of primary human

microvascular endothelial cells (pHMECs) by overexpression of the human telomerase catalytic

protein (hTERT). HMVECs have been extensively used in vitro angiogenesis studies (Yan et al., 2008;

Kobayashi et al., 2010; Ricci-Vitiani et al., 2010). These cells were kindly supplied by Dr. Xing Guo

(Duke University Medical Center, Durham, USA) and were cultured according to the protocol

described previously (Shao and Guo, 2004). Cells were used for our experiments until passage 20.

Since HMVEC is a pure cellular population there is no possible contamination by non-endothelial

cells.

Furthermore, all changes in gene expression need to be confirmed by Western

blotting.

We agree with the referee and we have now added a western blotting in Fig 1C. We also added

results concerning others endothelial and mesenchymal markers in Fig 1C. Actually, our results show

that Tie1 deficiency decreases the mRNA and protein expression of the vascular endothelial

markers(CD31, VE-Cad, CD34, FVIII), and increases expression of the mesenchymal markers (SMA,

S100A4, Col1a1, SM22N-cadherin) (Fig 1C). We also added new results concerning Tie2 deficiency.

Results show that Tie2 deficiency doesn’t modify the mRNA and protein expression of endothelial

(CD31, VE-Cad, CD34, FVIII) and mesenchymal markers (SMA, S100A4, Col1a1, SM22N-cadherin)

markers. These results have been added in Fig 1C.

Does Tie1 KD induce EC apoptosis?

Tie1 deficiency does not induce PARP cleavage that is one of the hallmarks of cell apoptosis. This

result is added in Additional data for the referees (Figure 2).

2. Supplementary Figure S1 only looks at cell morphology, cell

migration, ERK1/2 activity. How about smooth muscle markers, mesenchymal

markers, ERK5, Akt activity?

We thank the referee for this useful suggestion and performed these experiments. Results show that Tie1 deficiency in BAE cells strongly decreases the protein expression of the vascular endothelial

markers (CD31, VE-Cad, FVIII, CD34), and increases expression of the mesenchymal markers (SMA,

S100A4, N-cadherin, SM22Col1a1). We show that Tie1 deficiency in BAE cells induces Akt and ERK5 phosphorylation. These data are included in Supplementary Figure S1B and S1E respectively.

3.

4. The effect of Slug KG in Tie1 KD cells on gene expression is

totally unconvincing (with exception of collagen). Once again, the data is

presented very poorly.

All results are now presented with statistical tests demonstrating significant differences.

5. Since TGFbeta signaling is known to play a major role in End0-MT,

this signaling cascade needs evaluated after Tie1 KD.

This is a very interesting point and will be the subject of future studies. We plan to study the

interaction of other pathways involved in endothelial to mesenchymal transition not only TGF- signaling but also Notch and Wnt signaling pathways which are also strongly implicated in endothelial to mesenchymal transition (Potenta et al., 2008; Arciniegas, 2007).

6. Fig 3: the effect of Tie1 KD on cell migration is measured in the

PDGF stimulation assay. As normal EC express very little, if any, PDGF-R,

the differences in migration may be simply due to PDGF-R expression levels

that have not been assessed.

This is a very useful suggestion. Indeed the increase in migration with Tie1 deficiency can be explained by an increase in PDGF receptors: we have now performed these experiments and we show that Tie1 deficiency increases the mRNA and protein expression of the PDGFR-β. These results were added in the Fig 2D. The migration assay is not described but looks like a Boyden chamber assay.

Instead, the authors should examine unstimulated migration and migration in

response to VEGF and FGF (after first checking receptor levels before and

after Tie 1KD) in an in vitro wounding assay

We recognize that this migration test was very little described. We added a paragraph in the Methods section describing the migration test used and these words “in a modified Boyden chamber assay” in the legend to Fig 2D. We chose to use a directional test because PDGF is a chemotactic factor and PDGF-directional test is routinely used to assess migration (Ball et al., 2007; Veevers-Lowe et al., 2011). Concerning the migration in response to VEGF and FGF, this is a very interesting suggestion and will be the subject of future studies. 7. Fig 3 adhesion assay: why choose Fn as the matrix for this assay?

Several different matrices need to be assayed and expression of

corresponding integrins determined.

Our main objective was to evaluate the effect of PDGF on the migration of transformed endothelial cells. We chose fibronectin as matrix because this matrix is used in many publications concerning the chemotactic effect of PDGF (Ball et al., 2007; Veevers-Lowe et al., 2011). Fibronectin is a ubiquitous ECM protein and is a ligand for several integrins.

8. Fig 4: what time point is shown? In general, a time course is

required for these types of studies. Furthermore, Studies using high dose

(20 microM) chemical inhibitors are really not acceptable without

confirmation using dominant-negative constructs or something similar.

We apologize to not precisely described it in the legend to Fig 4 but only in the Methods section (RNA interference) and in Supplementary information. We have presently added this information in the legend to Fig 3 (late Fig4). We have added this sentence: “Cells were treated for 24h with MAPK (U0126 ; 10 µM) or/and Akt (LY-294,002 ; 10 µM) inhibitors” instead of “We used MAPK (U0126) and Akt (LY-294,002) inhibitors”. We agree with the reviewer that the 20 microM would be a high dose, but this was a mistake during the edition of the article; in fact we used the chemical inhibitors at the usual concentration of 10 microM (Tiwary et al, 2011; Bakin et al, 2001; Chatterjee et al. 2011) and not 20 microM. This point is now corrected in the revised version of the manuscript.

9. Fig 5: The authors need to show that Tie1 knockdown in EC inducea

Endo-MT in vivo. This is absolutely critical.

-Figure 5A and Figure 5C do not provide any useful information and should

be removed.

-We agree with the reviewer and remove Fig 5A because the coexpression of CD31+/SMA+ or CD31+/S100A4+ are represented in the triple-labeling experiments in the Fig 4A. -Li et al (Blood 2010) showed that human cells have 3 Tie-1 RNA

transcripts. Do all 3 transcripts increase in human pancreatic tumor +

metastasis samples? Are those RNA samples from whole tumors or isolated

primary EC from the metastasis tumors?

-The authors measure Tie-1 AS lncRNA expression by qPCR. What is the

specificity of this assay?

-In light of reviewer comment concerning the lncRNA analysis in pancreatic tumors, we decided to remove these experiments because we agree that these results are inconclusive and do not prove a mechanism for the inhibition of Tie1 in human pancreatic tumors.

-The authors state that Endo-MT is characterized by loss of CD31 in the

introduction and show that Tie-1 knockdown results in a loss of CD31 in EC

undergo Endo-MT. On page 8, the authors state that EC undergo Endo-MT have

both CD31 and FSP1/SMA markers. What gives?

This is exact but endothelial-mesenchymal transition is not a static phenomenon. The cells pass through one state to another with a series of changes. In our study, we characterized some of these changes, the disappearance of endothelial markers and the appearance of mesenchymal markers by in vitro experiments. In order to see the endothelial-mesenchymal transition in vivo, the only possibility is to detect the co-staining of endothelial and mesenchymal markers in the same cell. The labeling of different cells, one type with fibroblast markers and the other type with endothelial markers, would simply sign the existence of two different tissues into the sample.

-the authors use the term "complete phenotype switch" to characterize the

CD31-/SMA+ or CD31+/S100A4 cell population as cells undergo Endo-MT.

Without EC fate mapping, how do the authors know the cells are EC,

especially since Tie-1 and Tie-2 are not specific EC markers? Fate mapping

is absolutely required for End0-MT studies in vivo.

We agree with the reviewer and have changed the text accordingly. We describe a cell population which expresses both endothelial and mesenchymal markers and we speculate that this population could derive from an endothelial-mesenchymal transition. Fate mapping has been already described in another study (Zeisberg et al, 2007) in which these authors showed that, in endothelial cell–specific LacZ reporter mice, tumors contain fibroblasts that are also LacZ, thereby demonstrating their endothelial origin.

Minor

1. All figures are unnumbered which makes it rather hard on the

reader.

We apologize and have presently included a number in Figures. 2. Fig 1C is very poorly put together

We modified the Fig 1C as suggested.

3. Fig 2A presentation is very poor. It looks like there is a massive

increase in Snail expression until one notices the scale.

We have modified the Fig 2A as suggested. 4. The authors use HMVECs cells: what is the purity and passage

number of those cells? What is the source, purity, and passage number of

the BAE cells?

We used HMVECs which is an established cell line isolated after immortalization of primary human

microvascular endothelial cells (pHMECs) by overexpression of the human telomerase catalytic

protein (hTERT). HMVECs have been extensively used in vitro angiogenesis studies (Yan et al., 2008;

Kobayashi et al., 2010; Ricci-Vitiani et al., 2010). These cells were kindly supplied by Dr. Xing Guo

(Duke University Medical Center, Durham, USA) and were cultured according to the protocol

described previously (Shao and Guo, 2004). Cells were used for our experiments until passage 20.

Since HMVEC is a pure cellular population there is no possible contamination by non-endothelial

cells.

We thank to the reviewer for this comment because we forgot to give the characteristics of BAE cells.

We have added these sentences in the text of the section Supplementary Information: “Bovine Aortic

Endothelial (BAE) cells were kindly supplied by Dr. H. Drexler (Max Planck Institute, Bad Nauheim,

Germany). BAE cells were cultured in DMEM 1 g/l Glucose (Gibco) and 5% FBS. Only cells from

passage 12-16 were used in experiments.

5. Figure 5B and Figure 5C descriptions in text do not match the

figure legend.

This is also exact. We have made corrections accordingly.

References

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Additional data for the referees Figure 1: Specificity on Erk5 antibody. HMVECs were transfected twice with Erk5 or control (Ctrl) siRNA. Protein level was quantified by western blotting.

ERK5115 kDa

siRNA Ctrl Tie1

Figure 2: Tie1 deficiency does not induce PARP cleavage. HMVECs were depleted without serum or transfected twice with Tie1 or control (Ctrl) siRNA. Protein level was quantified by western blotting.

116 kDa

89 kDa

PARP

Cleaved

PARP

siRNA Depleted Ctrl Tie1ctrl

EMBO reports - Peer Review Process File - EMBOR-2011-35207

© European Molecular Biology Organization

2nd Editorial Decision 02 December 2011

Thank you for the submission of your revised manuscript to our journal. We have now received the full set of reports, copied below, from the two referees that were asked to re-evaluate it. As you will see, although referee #1 is very positive, referee #2 recommends rejection of your manuscript on the basis of lack of physiological relevance. After discussing his arguments with former referee #3, we have decided that fully addressing the role of Tie1 in vivo - albeit obviously interesting - is out of the scope of this paper. However, we think that, as referee #2 also proposes, at least some key experiments should be repeated in primary endothelial cells. I would particularly recommend to reassess the roles of Tie1 KD in EndMT (figure 1) and cell motility (figure 2). That, together with the already available information, should be sufficient to warrant publication of your study. Do not hesitate to contact me if you need any further information. I look forward to seeing a revised version of your manuscript when it is ready. Yours sincerely, Editor EMBO reports REFEREE REPORTS: Referee #1: The manuscript is now much improved. I think that it is acceptable for publication and that it is suitable for EMBO Reports. However, it would be much strengthened if the co-immunostaining analyses were quantified because it is now apparent that the number of cases analyzed is rather small. This does not detract from the primary interest of the work. Additionally, there is some need for editing of the text. Referee #2: I appreciate the effort that went into this revision of the manuscript. Unfortunately, critical issues remain. 1. The biggest problem is the authors used an immortalized cell line (HMVEC) to study EndMT. I actually did not realize in the original review that this was a cell line, and nor primary cells. Unfortunately, I see absolutely no way how an immortalized cell line may provide any meaningful information with regard to this process. How do we know that all of the observed is not a result of immortalization? At the very least experiments should have been repeated in primary endothelial cells. 2. One specific mechanistic issue that was raised was the role of TGF. This is not a trivial issue as there are half-a-dozen published EndMT papers that implicate TGF. Obviously, any new mechanism needs to be compared to the published one. 3. The biggest weakness is the very unconvincing, and minimal, in vivo data. The authors were asked to use endothelial fate mapped mice to show EndMT in their tumor model. This is not a complicated experiment. Unfortunately, this was not done. On the balance, the entire story is based on studies in an immortalized cell line with minimal and poor quality in vivo data.

EMBO reports - Peer Review Process File - EMBOR-2011-35207

© European Molecular Biology Organization

2nd Revision - authors' response 13 February 2012

Thankyouverymuchforyourreplyofourpaperentitled:

Tie1DeficiencyInducesEndothelial‐Mesenchymal‐Transition

by Julie Garcia, Maria José Sandi, Pierre Cordelier, Bernard Binétruy, Jacques

Pouysségur,JuanLucioIovannaandRoselyneTournaire

WehavenowconfirmedourresultsinprimaryHMVECcells.

WethereforerepeatedthekeyexperimentsthatyouhaverequestedinprimaryHMVEC

cells (Invitrogen). We obtained the same results as those obtained with immortalized

endothelial cells and primary BAE cells. We show that Tie1 deficiency induces a

morphological change and strongly decreases the protein expression of the vascular

endothelial markers (CD31, VE‐Cad, CD34, FVIII), and increases expression of the

mesenchymalmarkers (αSMA, S100A4, Col1a1, SM22α, N‐cadherin) in primary HMVEC

cells (Fig 1), in HMVEC cell line (Supplementary Fig S1) and in primary BAE cells

(Supplementary Fig S2). In addition, Tie1 siRNA increases the migration of primary

HMVEC cells (Fig 2D), HMVEC cell line (Supplementary Fig S3A) and primary BAE cells

(SupplementaryFigS2C).

Wethinkthattheseresultsshouldansweryourquestion.

3rd Editorial Decision 14 February 2012

I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to EMBO reports and congratulations on a successful publication. Please consider us again in the future for your most exciting work. Yours sincerely, Editor EMBO Reports