Homeobox A4 suppresses vascular remodeling by repressing YAP/TEAD transcriptional activity

Abstract The Hippo signaling pathway is involved in the pathophysiology of various cardiovascular diseases. Yes‐associated protein (YAP) and transcriptional enhancer activator domain (TEAD) transcriptional factors, the main transcriptional complex of the Hippo pathway, were recently identified as modulators of phenotypic switching of vascular smooth muscle cells (VSMCs). However, the intrinsic regulator of YAP/TEAD‐mediated gene expressions involved in vascular pathophysiology remains to be elucidated. Here, we identified Homeobox A4 (HOXA4) as a potent repressor of YAP/TEAD transcriptional activity using lentiviral shRNA screen. Mechanistically, HOXA4 interacts with TEADs and attenuates YAP/TEAD‐mediated transcription by competing with YAP for TEAD binding. We also clarified that the expression of HOXA4 is relatively abundant in the vasculature, especially in VSMCs. In vitro experiments in human VSMCs showed HOXA4 maintains the differentiation state of VSMCs via inhibition of YAP/TEAD‐induced phenotypic switching. We generated Hoxa4‐deficient mice and confirmed the downregulation of smooth muscle‐specific contractile genes and the exacerbation of vascular remodeling after carotid artery ligation in vivo. Our results demonstrate that HOXA4 is a repressor of VSMC phenotypic switching by inhibiting YAP/TEAD‐mediated transcription.

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Referee #1:
The manuscript by Masahiro Kimura and colleagues describes HoxA4-initially identified in an unbiased shRNA screen-as a putative repressor of Yap/Taz-TEAD transcriptional activity in vascular remodeling. In in vitro experiments in HEK293T and primary VSMC and in in vivo mouse arterial injury models, the authors provide evidence that HoxA suppression promotes cellular proliferation, YT-TEAD target gene activation and exacerbates vascular remodeling after carotid artery ligation. Vice versa, loss of HoxA4 or YAP-TEAD activation inhibits contractile smooth muscle marker gene expression.
Hox genes encode a subset of the homeobox transcription factors that are known to control the specification of anteroposterior identity in the animal embryo. They are also known to regulate stem cell differentiation and are usually dysregulated in cancer. Whereas the general role of YAP in phenotypic switching in VSMC has been suggested before, the negative regulation of this process by Hox4a and the proposed competitive inhibition of YAP binding to TEAD are to this reviewers knowledge novel and worth reporting in EMBO reports. Having said that, the manuscript although shedding light on HoxA as a putative negative regulator of TEAD singling, falls short in comprehensive experimental preparation and final conclusions on vascular remodelling in its current form. The following points should be addressed before publication: -The list/table of shRNAs that repress 8xGTIIC-EmGFP expression in different conditions "low and high density" and to which extend should be provided. They stated that they excluded shRNAs targeting ribosome biogenesis or transcription related genes that were enriched in the GFP high population, on the other hand they decided to have a closer look onto the function of HoxA4.
-Does HoxA4 have an effect on GTIIC or target gene expression in the presence of Yap-S94A (nuclear YAP lacking TEAD binding)? - Fig.3: title and conclusion too far-fetched "HoxA4 associates with TEADs to inhibit YAP-mediated transcriptional activation" The authors have shown immunoprecipitation studies of truncated TEAD1 and HoxA4 to map the minimal TEAD-HoxA4 interaction domain but lack experiments showing which truncations block YAP-Tead complex formation and GTIIC-dependent repression - Fig.4A: CHIP experiments for SMA, SM22a, Calponin and YT-target (e.g. Cyr61, CTGF) promotors using TEAD1/2, YAP and HoxA4 antibodies should be included preferably in VSMCs (TEAD4 is not expressed in VSMC) - Fig.4B: The authors stated that YAP and HoxA compete for binding to TEADs to inhibit YT target gene expression. They provided CHIP assay that point towards this conclusion, while the data for the competitive CoIP are not conclusive since also the amount of YAP-S127A in the input is reduced to the same extend. The question of whether shHoxA4 increases TEAD-YAP interaction needs also to be addressed. Quantifications are required and the IgG control in this particular experiment is missing. In general input controls for immunoprecipitation experiments throughout the manuscript lack Actin or Gapdh as loading control. - Fig.7: HoxA4-KO mouse and the arterial injury model Phenotype of HoxA4-ko mice: is there overgrowth in some organs were YT-TEAD signature is increased (eg lungs) and are other Hox family members overtaking the function of HoxA4 in HoxA4-KO? Immunofluorescence analysis, WB as well as qRT PCRs in carotid arteries of ctr and HoxA4-KO before and after ligation (or Right-nonligated, left-ligated)¬¬¬¬ should be provided to show the increase in YT-target gene expression and/or nuclear localization of YAP/TAZ before and after ligation.
-EVF2D: higher magnification needs to be provided as well as YAP and DAPI staining to show were HoxA4 and YAP localize in the different conditions -EVF5D: the author state that "VSMCs with nuclear HoxA4 were spindle shaped like a contractile phenotype, whereas cells without nuclear HoxA4 were rhomboid shaped like synthetic phenotype"(P12, lines 4-6). First, this is not obvious by the provided magnification it could be also dependent on the confluency. S¬¬¬¬econd, which cells are those , i.e. under which conditions HoxA4 can be found in the cytoplasm and does HoxA4 have a function there?

Referee #2:
The manuscript entitled 'Homeobox A4 suppresses vascular remodelling as a novel regulator of YAP/TEAD transcriptional activity' by Kimura et al., describes the role of HoxA4 as a novel suppressor of YAP/TEAD transcriptional activity as well as its role as a suppressor in the phenotypic switching of VSMCs. The authors have performed a thorough investigation to elucidate the molecular function of HOXA4 with proper validation. The findings are interesting for the field of YAP/ TEAD and transcriptional biology. However, no evidence of the interaction of HOXA4 and TEADs leading to attenuation of YAP activity is shown in VSMCs. Furthermore, a better characterization of the HOXA4 KO mice should be provided.
Major comments: 1. The key mechanistic findings of the study should be shown in VSMCs as well and not only in HEK293T.
2. Figure 2: the authors claim that the interaction between HOXA4 and TEAD would play a role during vascular remodeling; however, this interaction should be shown in VSMCs as well; and without overexpression of TEAD and HOXA4.
3. Figure 5G,H: counting the cell number does not really tell whether cells proliferate more or less, as other mechanisms such as cell death could be taking place. Therefore, this analysis should be replaced by a better proliferation assay (i.e. like BrdU labelling). Figure 5 (extended view): HOXA4 expression should be shown in vivo via immunostaining on tissue sections and not just based on the FANTOM5 CAGE data. 5. Figure 7: A better characterization of the HOXA4 KO mouse and in-vivo analysis needs to be shown. A staining for vSMCs and ECs would be a nice approach to show in general how vessels and SMCs look like in control and mutant embryos. 6. Could the contractibility of vSMC in control and HOXA4 mutants be measured? For example, in aortic rings? 7. Figure 7B : Complementary to Panel B: In order to link comprehensively the mechanism of how HOXA4 displaces YAP, does vascular remodeling induce HOXA4 expression? which would then displace YAP in vivo, as seen in the competition assay in vitro? A staining for HOXA4 could complement the RNA expression levels after artery ligation.

4.
8. Figure 7D: -Include H&E and/or Elastica van Gieson images from the four groups: right non-ligated artery WT animals; left ligated artery WT animals; right non-ligated artery HOXA4KO; left ligated artery HOXA4KO to have a better idea on how the neointima looks in every condition with the same staining.
-Include analysis of neointima area and neointima/media ratio for the four groups mentioned before.
-Complement the morphometric analysis with the other vessel parameters: vessel area and media area between the four groups.
Minor Comments: 1. A little work on wording and structure would make manuscript sound much better. Figure 3 and figure 4B, no 'n' number is provided.

2.
3. The authors have used student T-test in most of their analysis (comparison between 2 different variables), however, Mann whitney U test (non-parametric) was used in figure 7B. Is there a special reason for this? 4. The authors mention that "HOXA4 in vivo seems to be dispensable in steady-state conditions in adults". However, this is not shown in their study. Has this been properly investigated in VSMCs in vivo in other studies? Could the authors elaborate this more in discussion and give the appropriate references? 5. Figure 4B: It doesn't look like a dose response when expressing different levels of HOXA4. Qunatification of WB should be provided. 6. Figure 5E: contrast in aSMA and Calponin bands seems very high and artificial. Improve blot.  Figure 2 C and D: a one-way ANOVA was performed to determine statistical differences among groups however it is not stated whether control group (YAP5SA4 -, HoxA4-) has a significant difference with nuclear YAP overexpression (YAP5SA4 +, HoxA4-), especially in the analysis of the YAP target genes CTGF and CYR61.

A clearer labeling of Figure7 panels is recommended
1st Revision -authors' response 5 December 2019

Response to Referee #1
We are grateful to Referee #1 for the informative and useful comments. As described below, we have considered all of these comments and used them to improve our manuscript.

Hox genes encode a subset of the homeobox transcription factors that are known to control the specification of anteroposterior identity in the animal embryo. They are also known to regulate stem cell differentiation and are usually dysregulated in cancer. Whereas the general role of YAP in phenotypic switching in VSMC has been suggested before, the negative regulation of this process by Hox4a and the proposed competitive inhibition of YAP binding to TEAD are to this reviewers knowledge novel and worth reporting in EMBO reports. Having said that, the manuscript although shedding light on HoxA as a putative negative regulator of TEAD singling, falls short in comprehensive experimental preparation and final conclusions on vascular remodeling in its current form. The following points should be addressed before publication:
We thank the reviewer for the valuable and constructive comments on our manuscript. We have followed the suggestions and believe that these changes have considerably improved our manuscript

The list/table of shRNAs that repress 8xGTIIC-EmGFP expression in different conditions "low and high density" and to which extend should be provided. They stated that they excluded shRNAs targeting ribosome biogenesis or transcription related genes that were enriched in the GFP high population, on the other hand they decided to have a closer look onto the function of HoxA4.
As the reviewer suggested, we are also interested in shRNAs that repress TEAD-mediated transcriptional activity. Cells

Fig.3: title and conclusion too far-fetched. "HoxA4 associates with TEADs to inhibit YAP-mediated transcriptional activation" The authors have shown immunoprecipitation studies of truncated TEAD1 and HoxA4 to map the minimal TEAD-HoxA4 interaction domain but lack experiments showing which truncations block YAP-Tead complex formation and GTIIC-dependent repression.
We agree with the reviewer's comment. We modified the title as "Protein-protein interactions between HoxA4 and TEADs". To address this comment, we assessed the expression of target genes induced by YAP-5SA with each HOXA4-truncated mutant. The mutants that retained TEAD-binding homeodomains, HOXA4 201-320 or 216-272, significantly but only modestly suppressed their expression, implying a region other than the structural TEAD-binding sites of HOXA4 are necessary for YAP inhibition (Fig EV 3D). We added Figure EV3D and an explanation on page 10, lines 18-20, and modified the title of

Fig.4A: CHIP experiments for SMA, SM22a, Calponin and YT-target (e.g. Cyr61, CTGF) promotors using TEAD1/2, YAP and HoxA4 antibodies should be included preferably in VSMCs (TEAD4 is not expressed in VSMC).
We thank the reviewer for this essential and important comment. We performed ChIP assays using human vascular smooth muscle cells with the expression of tagged-HOXA4 and YAP-5SA (a nuclear-localized mutant), because we have no commercially available antibody against HOXA4 for ChIP, and YAP-DNA binding via TEADs is largely affected by YAP subcellular localization. As shown in Fig 6D, Figure 6D and an explanation on page 13, lines 3-7.
Inserted sentence (on page 13, lines 3-7): Disruption of the TEAD-HOXA4 interaction in VSMCs by knockdown of HOXA4 significantly increased the amount of endogenous TEAD-YAP complexes ( Fig 6C) and increased the occupation of HOXA4 on the TEAD-binding region in the promoters of CTGF and CYR61 but not αSMA attenuated that of phosphorylation-defective YAP (Fig 6D).

Fig.4B: The authors stated that YAP and HoxA compete for binding to TEADs to inhibit YT target gene expression. They provided CHIP assay that point towards this conclusion, while the data for the competitive CoIP are not conclusive since also the amount of YAP-S127A in the input is reduced to the same extend. The question of whether shHoxA4 increases TEAD-YAP interaction needs also to be addressed. Quantifications are required and the IgG control in this particular experiment is missing. In general input controls for immunoprecipitation experiments throughout the manuscript lack Actin or Gapdh as loading control.
We conducted CoIP assays again in HEK 293T and showed that the TEAD-YAP interaction was inhibited by HOXA4 in a dose dependent manner by quantification of protein, and a Gapdh loading control and IgG IP-control were also provided ( Fig 4B). We have also shown that the TEAD-YAP interaction was increased by endogenous HOXA4 knockdown in vascular smooth muscle cells ( Fig  6C). We modified Figure 4B, and added Figure 6C and an explanation on page 13, lines 3-7.
Inserted sentence (on page 13, lines 3-7): Disruption of the TEAD-HOXA4 interaction in VSMCs by knockdown of HOXA4 significantly increased the amount of endogenous TEAD-YAP complexes ( Fig 6C) and increased the occupation of HOXA4 on the TEAD-binding region in the promoters of CTGF and CYR61 but not αSMA attenuated that of phosphorylation-defective YAP (Fig 6D).

Fig.7: HoxA4-KO mouse and the arterial injury model Phenotype of HoxA4-ko mice: is there overgrowth in some organs were YT-TEAD signature is increased (eg lungs) and are other Hox family members overtaking the function of HoxA4 in HoxA4-KO?
We thank the reviewer for this comment. We measured several organ weights, including the lung; however, we did not find any overgrowth of these organs (Appendix Fig S6A). Recently (Horan et al, 1994). In this study, we found Hoxa4 KO mice showed exacerbated neointima formation of the ligated carotid artery, in which YAP/TEAD transcriptional activity is highly upregulated (Fig 6). On the other hand, we found Hoxa4 KO mice showed exacerbated neointima formation of the ligated carotid artery, in which YAP/TEAD transcriptional activity is highly upregulated (Fig 7).  (Fig 4A).

Immunofluorescence analysis, WB as well as qRT PCRs in carotid arteries of ctr and HoxA4-KO before and after ligation (or Right-nonligated, left-ligated) should be provided to show the increase in YT-target gene expression and/or nuclear localization of YAP/TAZ before and after ligation.
We thank the reviewer for this very constructive comment. We presented immunofluorescence analysis ( Fig EV5A) and WB (Fig 7F) in non-ligated and ligated carotid arteries, and confirmed the upregulation of YT-target genes as well as the downregulation of smooth muscle contractile genes in Hoxa4-deficient mice compared with WT mice, in accordance with the results of qRT-PCR. Furthermore, qRT-PCR analysis showed that differences in YT-target genes between control and Hoxa4 KO mice were also significant after ligation (Fig 7E). We added Figure 7E, 7F, EV5A and explanations on page 14, lines 17-22. Inserted sentences (on page 14, lines [17][18][19][20][21][22]: The predominant expression of YAP/TEAD target genes in the carotid arteries of Hoxa4 KO mice was also observed at 1 week after ligation (Fig 7E). These differences in differentiation-or proliferation-associated genes in the injured artery between WT and Hoax4 KO mice were also confirmed by immunoblotting (Fig 7F) or immunohistochemistry (Fig EV5A), leading to a two-fold increase in neointima formation in Hoxa4 KO mice at 250µm proximal to the ligation (Fig 7G to 7I) and at 500µm proximal to the ligation (Fig EV5B to 5D).

EVF2D: higher magnification needs to be provided as well as YAP and DAPI staining to show were HoxA4 and YAP localize in the different conditions.
We showed immunostaining of HEK 293T cells transfected with HOXA4-GFP using DAPI, anti-GFP and anti-YAP antibodies (Fig EV1E), and also GFP fluorescence with higher magnification (Fig EV1D) with different cell densities. These data suggested that HOXA4 expression did not affect YAP subcellular localization regulated by the Hippo signaling pathway. We added Figure EV1D, 1E and an explanation on page 8, line 22 -page 9, line 2.

Inserted sentence (on page 8, line 22 -page 9, line 2):
Because HOXA4 persistently stays in the nucleus without affecting YAP subcellular localization (Fig EV1D and 1E), we speculated that HOXA4 might physically interact with TEADs or YAP.

EVF5D: the author state that "VSMCs with nuclear HoxA4 were spindle shaped like a contractile phenotype, whereas cells without nuclear HoxA4 were rhomboid shaped like synthetic phenotype"(P12, lines 4-6). First, this is not obvious by the provided magnification it could be also dependent on the confluency. Second, which cells are those, i.e. under which conditions HoxA4 can be found in the cytoplasm and does HoxA4 have a function there?
We re-assessed HOXA4 subcellular localization in human vascular smooth muscle cells by immunostaining; however, HOXA4 existed only in the nucleus with a some reproducibility. Therefore, we modified the images (Fig EV4E) and concluded that HOXA4 constitutively stays in the nucleus. We modified Figure EV4E and deleted the sentence below.
Deleted sentence: Of note, VSMCs with nuclear HOXA4 was spindle shaped like a contractile phenotype, whereas cells without nuclear HOXA4 was rhomboid shaped like synthetic phenotype (Fig EV5D).

Response to Referee #2
We are grateful to Referee #2 for the informative and useful comments. As described below, we have considered all of these comments and used them to improve our manuscript.

The manuscript entitled 'Homeobox A4 suppresses vascular remodeling as a novel regulator of YAP/TEAD transcriptional activity' by Kimura et al., describes the role of HoxA4 as a novel suppressor of YAP/TEAD transcriptional activity as well as its role as a suppressor in the phenotypic switching of VSMCs. The authors have performed a thorough investigation to elucidate the molecular function of HOXA4 with proper validation. The findings are interesting for the field of YAP/ TEAD and transcriptional biology. However, no evidence of the interaction of HOXA4 and TEADs leading to attenuation of YAP activity is shown in VSMCs. Furthermore, a better characterization of the HOXA4 KO mice should be provided.
We agree and thank the reviewer for the excellent and constructive comments, which have helped to considerably improve our manuscript. -Major

The key mechanistic findings of the study should be shown in VSMCs as well and not only in HEK293T.
We strongly agree with the reviewer's comment, which was also pointed by the other reviewer. We also performed competitive co-IP assays and ChIP assays using human vascular smooth muscle cells and obtained consistent results with those seen in HEK 293T cells (Fig 6C and 6D). We added Figure 6C, 6D and explanations on page 13, lines 3-7. 6C) and increased the occupation of HOXA4 on the TEAD-binding region in the promoters of CTGF and CYR61 but not αSMA attenuated that of phosphorylation-defective YAP (Fig 6D).

Figure 2: the authors claim that the interaction between HOXA4 and TEAD would play a role during vascular remodeling; however, this interaction should be shown in VSMCs as well; and without overexpression of TEAD and HOXA4.
We strongly agree with the reviewer's comment, which was also pointed by the other reviewer. We proved that endogenous knockdown of HOXA4 increased TEAD-YAP binding in vascular smooth muscle cells using a co-IP assay (Fig 6C).

Figure 5G, H: counting the cell number does not really tell whether cells proliferate more or less, as other mechanisms such as cell death could be taking place. Therefore, this analysis should be replaced by a better proliferation assay (i.e. like BrdU labelling).
We agree with the reviewer on this comment. We conducted a BrdU incorporation assay in vascular smooth muscle cells with overexpression or knockdown of YAP or HOXA4, and the results were consistent with the cell count assay (Fig 5H, 5J, EV4F and EV4G). We added Figure 5H, 5J, EV4F, EV4G and an explanation on page 12, lines 13-15.

Inserted sentence (on page 12, lines 13-15):
This negative effect of HOXA4 on the proliferative capacity of VSMCs was also confirmed by bromodeoxyuridine (BrdU) incorporation analysis (Fig 5H and 5J).

Figure 5 (extended view): HOXA4 expression should be shown in vivo via immunostaining on tissue sections and not just based on the FANTOM5 CAGE data.
We first tried to perform Hoxa4 immunostaining on mouse tissue sections. Although we tried all commercially available antibodies against mouse Hoxa4 (described in the Methods), we found no specific antibodies against mouse Hoxa4, because several bands or non-specific staining were still observed even in Hoxa4-deficient mice. Meanwhile, we could certainly detect human HOXA4 protein, which was confirmed using both knockdown and overexpression experiments. Therefore, human aortic samples were used to confirm the expression of HOXA4 in vascular smooth muscle cells in vivo (Fig EV4B). We added Figure EV4B and an explanation on page 11, lines 10-11.
Inserted sentence (on page 11, lines 10-11) : We also confirmed the expression of HOXA4 in a human aortic tissue (Fig EV4B).

Figure 7: A better characterization of the HOXA4 KO mouse and in-vivo analysis needs to be shown. A staining for vSMCs and ECs would be a nice approach to show in general how vessels and SMCs look like in control and mutant embryos.
We performed immunostaining on dorsal aorta of E18.5 mouse embryos to compare Hoxa4 KO with WT mice, which showed no difference in the staining pattern of vascular smooth muscle cells and endothelial cells (Appendix Fig S6B). We added Appendix Figure S6B and an explanation on page 13, lines 19-21. Inserted sentence (on page 13, lines [19][20][21]: The contractibility of primary VSMCs harvested from Hoxa4 KO and WT mice was also compared using a collagen gel assay and found to be almost similar (Appendix Fig S6B).

Could the contractibility of vSMC in control and HOXA4 mutants be measured? For example, in aortic rings?
We conducted collagen gel contraction assays to assess contractibility using primary aortic vascular smooth muscle cells harvested from WT and Hoxa4 KO mice, which showed no difference (Appendix Fig S6C). We added Appendix Figure S6C and an explanation on page 13, line 21 to page 14, lines 1-2.
Inserted sentence (on page 13, line 21 to page 14, lines 1-2): The contractibility of primary VSMCs harvested from Hoxa4 KO and WT mice was also compared using a collagen gel assay and found to be almost similar (Appendix Fig S6B).

Figure 7B : Complementary to Panel B: In order to link comprehensively the mechanism of how HOXA4 displaces YAP, does vascular remodeling induce HOXA4 expression? which would then displace YAP in vivo, as seen in the competition assay in vitro? A staining for HOXA4 could complement the RNA expression levels after artery ligation.
We fully agree on the importance of how endogenous Hoxa4 protein expression changes after vascular injury, and hoped to make it clear. Unfortunately, we could not find any specific anti-Hoxa4 antibody for immunostaining on mouse tissues, although we tried all commercially available antibodies. There is only one report showing a reduction in HOXA4 comparing aortic smooth muscle cells of an aortic aneurysm with those of normal aorta by immunostaining ( In order to exclude the possibility that a functional truncated peptide was translated, we deleted almost all genomic regions of Hoxa4 by inducing double-strand breaks at two sites around the start and stop codons and following homologous recombination repair (Appendix Fig S5B and 5C).
Inserted sentence (on page 14, lines 14-16): Although we could not detect a specific signal of mouse Hoxa4 protein due to the absence of commercially available antibodies (See Methods), the transcription of Hoxa4 in the ligated carotid artery was significantly reduced at 1 week after operation (Fig 7C).

Figure 7D: -Include H&E and/or Elastica van Gieson images from the four groups: right non-ligated artery WT animals; left ligated artery WT animals; right non-ligated artery HOXA4KO; left ligated artery HOXA4KO to have a better idea on how the neointima looks in every condition with the same staining. -Include analysis of neointima area and neointima/media ratio for the four groups mentioned before. -Complement the morphometric analysis with the other vessel parameters: vessel area and media area between the four groups.
We agree with the reviewer on this comment. As indicated by the reviewer, we added Figure 7G to 7I, Figure EV5B to EV5D, and modified an explanation on page 14, lines 18-22.
Modified sentence (on page 13, lines 14-17): These differences in differentiation-or proliferation-associated genes in the injured artery between WT and Hoax4 KO mice were also confirmed by immunoblotting (Fig 7F) or immunohistochemistry (Fig EV5A), leading to a two-fold increase in neointima formation in Hoxa4 KO mice at 250µm proximal to the ligation (Fig 7G to 7I) and at 500µm proximal to the ligation (Fig EV5B to 5D). -Minor

A little work on wording and structure would make manuscript sound much better.
We thank the reviewer for this advice. We have tried to improve the manuscript structure.

Figure 3 and figure 4B, no 'n' number is provided.
We

The authors have used student T-test in most of their analysis (comparison between 2 different variables), however, Mann whitney U test (non-parametric) was used in figure 7B. Is there a special reason for this?
There was no special reason for this; however, groups with similar variance were analyzed using parametric tests, and groups with significantly different variance were analyzed using nonparametric tests.

The authors mention that "HOXA4 in vivo seems to be dispensable in steady-state conditions in adults". However, this is not shown in their study. Has this been properly investigated in VSMCs in vivo in other studies? Could the authors elaborate this more in discussion and give the appropriate references?
We thank the reviewer for raising this point. The molecular function of HOXA4 in VSMCs has not been investigated previously and we have no data to conclude this; therefore, we have removed this sentence in the Discussion section from the manuscript.
Deleted sentence: HOXA4 in vivo seems to be dispensable in steady-state conditions in adults

Figure 4B: It doesn't look like a dose response when expressing different levels of HOXA4. Qunatification of WB should be provided.
We thank the reviewer for this comment, which was also pointed by the other reviewer. We conducted the competitive co-IP assays again and confirmed a dose-responsive decrease in TEAD-YAP binding using different levels of TEAD-HOXA4 binding with the same amount of input YAP. Quantification of immunoblotting is also shown in Fig 4B. We modified Figure 4B.
6. Figure 5E: contrast in aSMA and Calponin bands seems very high and artificial. Improve blot.
We have shown the improved images. We modified Figure 5E.

Typo on expanded view figure 4, panel B: should say fold CHANGE
We have corrected the manuscript accordingly. We modified Figure 4B.

Typo on expanded view figure 5, panel B: should say RELATIVE
We have corrected the manuscript accordingly. We modified Figure 5B.

Figure 2 C and D: a one-way ANOVA was performed to determine statistical differences among groups however it is not stated whether control group (YAP5SA4 -, HoxA4-) has a significant difference with nuclear YAP overexpression (YAP5SA4 +, HoxA4-), especially in the analysis of the YAP target genes CTGF and CYR61.
We have added the mark to indicate significance in the figure. We modified Figure 2C, 2D, EV1A, EV1B, EV1C, EV3B and EV3C.

A clearer labeling of Figure7 panels is recommended.
We have changed the labeling accordingly. We modified Figure 7A, 7B, 7D and 7E.
2nd Editorial Decision 22 January 2020 Thank you for submitting the revised version of your manuscript. It has now been seen by one of the original referees, whose comments have been pasted below. My apologies for this unusual delay in getting back to you. It took longer than anticipated to receive the referee report due to the recent holiday season.
As you can see, the referee finds that the study is significantly improved during revision and recommends publication here. Before I can accept the manuscript, I need you to address some minor points below: •

Referee #2:
The authors have address most of the comments raised by this reviewer. In its current state the manuscript has been improved and it has become a comprehensive study. This reviewer has no further comments or raised issues.
2nd Revision -authors' response 31 January 2020 The authors performed all minor editorial changes. 3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
For animal studies, include a statement about randomization even if no randomization was used.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe.

B-Statistics and general methods
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No statistical method was used to determin the sample size, but the experiment was carried out biological triplicate based on our previous studies. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
Sample size for animal studies was estimated by previous studies using simillar methodologies.
No sample and animals were excluded basically.Only one mouse in each group (WT and KO) was excluded for analysis of ligated carotid artery at 250 µm proximal to ligation because of the fragmentation of the vessels.
For mouse studies, 8-weeks-old male KO and WT littermates were used. Mice used for experiments were randomly selected from each group.

Manuscript Number: EMBOR-2019-48389V1
Yes, each statistic tests used are described in the figure legends.
We used Shapiro-Wilk normality test which is an in-built analysis of Graph Pad Prism.

None.
Analysis in murine carotid artery injury models and BrdU incorporation assays was performed by an experimenter who was blinded to treatment groups. None.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.