c-Src induced vascular malformations require localised matrix degradation at focal adhesions

  1. Patricia Essebier
  2. Lilian Schimmel
  3. Teodor Yordanov
  4. Mikaela Keyser
  5. Alexander Yu
  6. Ivar Noordstra
  7. Brittany Hill
  8. Alpha S. Yap
  9. Samantha J. Stehbens
  10. Anne K. Lagendijk
  11. Emma J. Gordon

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Abstract

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

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    Reply to the reviewers

    1. General Statements

    We appreciate the reviewers’ thoughtful feedback and thank them for their valuable suggestions to improve the manuscript. We have endeavored to respond to all their comments, with many of their concerns already incorporated in the manuscript. Validations for the additional experiments to be incorporated into the manuscript have been performed and show that all the plans outlined in Section 2 are highly feasible and will be added for the full revision. We believe that the incorporated and planned revisions contribute to a significant improvement on the original manuscript.

    2. Description of the planned revisions

    Reviewer 1

    Major comments:

    Point 3. p. 5. The authors do not describe any relationship to notch signaling. But notch signaling is the mechanism by which a sprout is selected. The CA phenotype shows no selection, and every sprout can continue migration. Did the authors check for any relationship between notch signaling c-Src activation? Does upregulation of C-Src downregulate notch?

    In previous unpublished results examining the impact of the loss of endothelial c-Src on notch signaling, we observed no alteration in DLL4 expression in the sprouting retina on postnatal day 5. Furthermore, no change in tip cell number was observed in mice with a loss of endothelial c-Src, suggesting c-Src depletion does not impact notch activity (Schimmel et al., Development, 2020, Figure 1M). We have started additional preliminary experiments performing immunostaining with a DLL4 antibody in migrating c-Src-CA cells to assess activation of notch signaling upon c-Src activation. We will continue these experiments for the full revision and will confirm the results via further analysis of notch activation by assessing DLL4 expression in the c-Src mutant cells using Western blot.

    Reviewer 2

    Major comments:

    Point 1. The authors have only used one type of vein endothelial cells from one single donor but they conclude that is effect is general for all endothelial cells. Endothelial cells are very heterogeneous, not only depending on their function and localization, vein, artery or capillary, but also between different organs and in disease (PMID: 22315715, PMID: 28775214, PMID: 31944177, PMID: 33514719). The authors, should either repeat some of the key experiments in other type of endothelial cells, maybe arterial or microvasculature cells which are commercially available or at least state that the observations presented in this manuscript apply to HUVECs and discuss whether this would also apply for other cell types.

    We agree it would be highly beneficial to assess whether c-Src-CA induces vascular expansion in other endothelial cell types. We have successfully transduced human arterial endothelial cells (HAEC) with empty vector and c-Src-CA lentivirus and are able to grow HAECs in 3D vessels. This demonstrates that introducing the c-Src constructs into other endothelial cells and putting them in 3D assays is highly feasible. We have also used human microvascular endothelial cells (HMVEC) in 3D vessels in previous studies (Schimmel et al., Clin Trans Immunol, 2021). Therefore, we will perform experiments introducing the full set of c-Src mutations in HAEC and/or HMVEC in 3D vessels for the revision to strengthen our findings.

    Reviewer 3

    *Major comments: *

    Point 1. "This was further supported by our observation that there were no changes in proliferation in c-Src mutant cells grown in a 2D monolayer". Figure 1A appears to have increased number of cells in the c-Src-CA condition compared to the control condition. Could the authors quantify the number of cells/area as they did for their 3D vessel model? This would reinforce the idea that the ballooning phenotype they observe is not due to differences in proliferation.

    We have started quantification on the number of cells per bead for the 3D bead sprouting experiments shown in Figure 1. We will complete this quantification for 3 independent experiments and the results will be added for the full revision.

    Point 2. Would be strengthened with analysis of another proliferation marker, such as EdU label, which is incorporated only during S phase of the cell cycle. Comparing ki67 staining and EdU staining would provide more insights. Also, using their 3D vessel model for this analysis would increase its relevance.

    We agree that showing proliferation in a 3D setting would be highly beneficial. We tested proliferation marker Ki67 in 3D vessels to ensure this analysis will be possible. We will perform full analysis of proliferation across c-Src mutations in 3D for the revision. We have started with BrdU labelling in 2D, and we will perform full analysis of proliferation with BrdU across c-Src mutations for the revision.

    Point 3. In Figure 1E', cells expressing the constitutively active form of cSrc appear to detach, giving the impression of cell death. Have the authors tested the viability/apoptosis of c-Src-CA cells, particularly in their 3D model?

    We agree that showing cell death in our model, especially in a 3D setting, would be highly beneficial. We have tested cell death marker Cleaved Caspase 3 (CC-3) in 3D vessels to ensure this analysis is feasible. We will perform full analysis of cell death across c-Src mutations in 3D for the revision.

    Point 4. "Therefore, reduction of endothelial cell-cell contacts in c-Src-CA cells may be due to elevated VE-cadherin phosphorylation and subsequent internalisation", "As reduction in cell-cell junction integrity has been shown to increase migratory capacity and sprouting angiogenesis [38], our data suggest that a balanced control of both cell-matrix and cell-cell junctions is essential for mediating migration." In general, it's not clear how constitutively active cSrc affects focal adhesions and cell-cell adhesion and how this is responsible for their ballooning phenotype. The role of the phosphorylation of the VE-Cadherin and cell-cell junctions in this process is not clear either. Further analysis of cell-cell junctions and focal adhesions (co-staining of phosphorylated paxillin and VE-Cadherin) and focal adhesions/fibronectin (like in figure 4C) in the context of cell migration (scratch wound assay) would provide important information to strengthen this notion of balanced control of both cell-matrix and cell-cell junctions.

    We will perform experiments on migrating cells in 2D, co-staining for p-paxillin and VE-cadherin, and p-paxillin and Fibronectin, to address the role of balanced cell-matrix and cell-cell junction adhesion, and how they influence Fibronectin deposition in migrating cells.

    Point 6. "Taken together, these results reveal that proteases produced by c-Src-CA cells are locally secreted at FAs but are membrane bound." The claim that proteases are membrane-bound is not convincingly demonstrated. Could the authors assess whether the constitutive form of cSrc activates the expression of specific genes encoding MMPs by qPCR? Or is it more a matter of the effect of c-Src on the transport of MMPs by microtubules?

    We would like to clarify the content of Figure 5, which presents two distinct sets of experiments supporting the assertion that the proteases under investigation are membrane-bound. Firstly, the transfer of conditioned medium from c-Src mutant cells demonstrated no degradation of fibronectin fibrils. Secondly, in the bead sprouting assay, a mixed culture of untransduced and c-Src-CA expressing cells was utilised. The results revealed that only c-Src-CA cells formed balloons, while untransduced cells sprouted normally right next to or sometimes even through a balloon.

    Recognising the need for a more in-depth understanding, we acknowledge the importance of analysing specific MMP gene expression. To this end, we have ordered qPCR primers for distinct MMPs, namely MMP2, MMP7, MMP9, and MT1-MMP. These forthcoming experiments are not only highly feasible but will also contribute valuable insights. The results of this gene expression analysis will be incorporated into the revision, shedding light on whether constitutively active c-Src induces MMP gene expression or influences MMP transport.

    Minor comments:

    Point 2. The lab already showed in a previous study that mice lacking c-Src specifically in endothelial cells have reduced blood vessel sprouting, leading to the expectation that the constitutively active form of cSrc would increase sprout number in the sprouting assay. Could the authors explain why the constitutively active form of cSrc induces this vascular ballooning and not an increase in the number of sprouts?

    In line with analysis to be performed on notch activity and DLL4 expression (Reviewer 1 point 3), we will provide additional discussion on the role of notch signalling and tip cell identity with the full revision.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    R____eviewer 1

    Major comments:

    Point 1. p5. Fig 1: The sentence that the dominant negative completely abrogated 'this' phenotype implies that the dominant negative was put into the same cells as the constitutively active mutation. 'Abrogated' means it stops the phenotype, and the phenotype in the sentence prior was constitutively active. It is more accurate to say that the dominant negative was not distinguishable from wild type, which is what the statistics show. No double transfection (DN-CA) was performed.

    We have changed the wording in the manuscript accordingly to ‘The c-Src-DN mutation showed no phenotype distinguishable from Ctrl (Fig 1A-D).’ on page 5.

    Point 2. p.5. Fig 1: the phenotype of the CA cells is fascinating. They expand far beyond their normal territory, but they are held together in a lacy bubble. To me, this looks like a different phenotype from the ballooning that might occur in an arteriovenous malformation in vivo, as in vivo malformations are continuously covered by cells. I understand why the authors might use the term ballooning but given that the cells expand without continuously touching each other, I do not think this is the correct term. Would blebbing, or radial migration in a lace-like discontinuous pattern describe it better?

    We have changed the phrasing from ‘ballooning morphology’ to ‘radial migration in a lace-like discontinuous pattern’ on page 5. For brevity, this has been referred to as ‘ballooning’ for the remainder of the manuscript, as noted on page 5.

    Point 4. The statistical methods are not described in the methods (GraphPad?). These need to be added. Are only significant comparisons plotted? In Fig 6 and 7 only pairwise statistics are shown. If all significant comparisons are plotted, then this means that the comparison between the rescued CA and the treated or untreated control is not significant. This can be thought of as a partial rescue towards a wild type, but it is definitely not a full rescue. None of the statistical comparisons in Figure 6 or 7 show significant comparisons to wildtype. This needs more discussion.

    We have now added additional clarification on statistical methods. Details on the statistical tests for each figure are mentioned in the figure legends. A general section on the statistical methods is now added to the methods section on page 18. Only significant comparisons are displayed in the graphs, but as mentioned by reviewer 2 (minor point 2), we have added additional information for transparency. Each of the different comparisons that were made, and their precise p value, have been compiled a table which has been added as Supplementary Table 1 to the manuscript.

    In Figures 6 and 7, we exclusively plotted pairwise comparisons to assess the impact of Marimastat treatment. As outlined in Supplementary Table 1, there is still a statistical significance when comparing Marimastat-treated c-Src-CA with either Marimastat-treated Ctrl or Marimastat-treated c-Src-WT. This suggests a partial rescue. For clarity, we kept only pairwise comparisons in the graphs, but discussed the partial rescue due to remaining significant difference between Marimastat-treated c-Src-CA and Ctrl or c-Src-WT cells in the results, referring to Supplementary Table 1 for p values. An important sidenote: c-Src-CA treated cells cannot exhibit complete rescue since they are initially seeded without Marimastat, and have already initiated ballooning by the time treatment commences.

    Point 5. Mmp activity is inferred, but not measured. This is a limitaion as the assumption is that marimostat acting through the expected pathway.

    Marimastat is one of the most commonly used broad spectrum MMP inhibitors, with potent activity against major MMPs, including MMP1, MMP3, MMP2, MMP9, MMP7 and MMP14. This is outlined in the existing reference (Rasmussen and McCann, 1997). We have adjusted phrasing to clarify the potency of Marimastat and have emphasised this is an MMP targeting drug which has been widely utilised in oncology clinical trials (page 8).

    Minor comments:

    Point 1. Fig 5D. The presentation of the data in this graph is difficult to understand. It is trying to show the proportion of mScarlet in sprouts or balloons a percentage of all the scarlet cells. It would be better to have all cells represented in one bar, distributed between sprout and balloon in that one bar. i.e., for the control and dominant negative, the bars would be all black and then for the CA it would be all white. The zero data points are confusing. A proportions graph should be investigated here.

    We have changed the graph in Figure 5D, which now represents the % of the outgrowth area, sprouts for Ctrl, c-Src-WT and c-Src-DN and balloon for c-Src-CA, that are mScarlet positive. Resulting in all black bars for Ctrl, c-Src-WT and c-Src-DN and all white bar for c-Src-CA, as the reviewer predicted.

    Point 2. The methods for vessel coverage for quantification in figs 1 and 7 are missing.

    We have added details of how quantification of vessel coverage in Figure 1 and 7 was performed to the methods section on page 17/18 as follow: ‘Microfluidic vessel coverage was measured by tracing any holes in the vessel wall (inverse of cell area marked by phalloidin) and dividing this by the total cell area per image.’

    Reviewer 2

    Minor comments:

    Point 1. Although the methods are well written and can be understood. To improve transparency, the authors should reduce the referring to other papers to describe the methods they perform and at least some kind of brief description should be included.

    We have added a brief description of the methods that included references to other papers; lentiviral transduction and microfluidic devices. More details about the lentivirus transduction were added on page 15 and a short description about the fabrication of the microfluidic devices was added on page 15/16.

    Point 2. The authors should report the real p value for their tests. Also, when the test is not significant.

    To provide more transparency about all of the different comparisons that were made and their precise p value, we have compiled a table listing all the p values and which is added as Supplementary Table 1 to the manuscript.

    Reviewer 3

    Minor comments:

    Point 3. In Figure 1A, it would be beneficial to include images from orthogonal views. Indeed, in the c-Src-CA condition, it's not clear whether the vascular ballooning observed represents a cluster of cells or an empty space between the bead and the endothelial cells. (Supp movie 1 helps, but it would be useful to add orthogonal views to the figure)

    For clarity, we have added single Z plane image for cross sectional views of the bead sprouts in Figure 1A to show that the c-Src-CA cells have an empty space inside the balloon, rather than being a big cluster of cells.

    Point 4. In Figure 1D, the method used to analyze sprout shape is not clear, especially for the c-Src-CA condition where the number of sprouts is close to 0. The figure legend indicates that this measurement corresponds to the shape of the sprouting area. Could the authors clarify and explain their quantification method?

    The shape of the sprouting area refers to the circularity index of the vascular area, measured by tracing the perimeter of the cell area in a minimum Z-projection of brightfield images and subtracting the area of the bead. For better clarity, we have adjusted the title of Figure 1D and Figure 6D to ‘Vascular area shape’ and added details of the quantification method in the methods section on page 17.

    Point 5. "however cells within the vessel still maintained some connections (Fig 1E')": The connections between cells are difficult to see in the images in Figure 1E'. Could the authors provide higher magnification images of the VE-cadherin staining to illustrate these connections between cells?

    For improved clarity, we have added high magnification images of the VE-cadherin channel only in black and white (Figure 1E’’) and indicated some of the maintained cell-cell connections in the c-Src-CA cells with black arrowheads.

    Point 6. "The reduction in migration correlated with an increase in FA size c-Src-CA expressing cells.": Could the authors give more explanation?

    We have adjusted phrasing to provide additional information (page 6/7) as follows: ‘The reduction in migration velocity in c-Src-CA cells coincides with an increase in FA size, number and density (Fig 2A-D). This suggests that the reduction of migration velocity is due to increased cellular adhesion via FAs.’

    Point 7. Could the authors widen the cell trajectory trace in Supplementary Figure 3A?

    We have adjusted the trajectory traces in Supplementary Figure 3A with wider lines for improved visibility.

    Point 8. it is very difficult to distinguish fibronectin fibrils on the images shown in figure 4C. it would be beneficial to change the images.

    We have enlarged the zoomed areas for better visibility of the focal adhesions and fibronectin degradation underneath those areas in the c-Src-CA cells. Additionally, arrows are added to indicate fibronectin fibrils.

    Point 9. "Treatment of ECs with Marimastat in a fibrin bead sprouting assay resulted in a rescue of the ballooning morphology observed in the c-Src-CA cells" Based on the images displayed in the figure and the associated quantifications, it still appears that c-Src-CA+Marimastat induces a vascular ballooning even if it is less pronounced than in the DMSO condition. Hence, it would be more accurate to describe the observed effect as a "partial rescue". In the microfabricated 3D vessel, in the figure 7A, cell-cell junctions still appear altered by c-Src-CA after the treatment with Marimastat, compared to the c-Src-WT-Marimastat, it would be more appropriate to talk about "partial rescue".

    We have changed ‘rescue’ to ‘partial rescue’ when referring to results in Figure 6 and 7 (page 8).

    Point 10. In Figure 6A, it seems that there is a decrease in the number of sprouts in the c-Src-DN condition compared to the control condition after the DMSO treatment, which is not observed in Figure 1, could the authors explain why?

    In Figure 1C, the number of sprouts is also reduced in the c-Src-DN condition compared to c-Src-WT, but this is not significant when compared to control (see Supplementary Table 1 for p values of all comparisons). However, it is true that the number of sprouts in the c-Src-DN condition is significantly reduced compared to both control and c-Src-WT upon DMSO treatment (Fig 6C). Reduction of sprouts in c-Src-DN cells was expected due to the dysfunctional kinase domain, as mentioned on page 5 and shown in reference 30 (Shvartsman, D.E., et al., J Cell Biol, 2007. 178(4): p. 675-86.). Why DMSO treatment seems to enhance the effects of dominant negative c-Src expression on sprouting behaviour remains unclear. However, DMSO has adverse effects on sprouting shown by reduction of sprouts in both control and c-Src-WT cells (comparing untreated condition in Fig 1C with DMSO treated condition in Fig 6C). We believe that DMSO treatment is an extra challenge for cells on top of c-Src-DN expression, which therefore display reduced sprouting compared to control and c-Src-WT.

    Point 11. There is no statistical paragraph in the method section.

    As pointed out by reviewer 1 and 2, we have now added a general section on the statistical methods to the method section on page 18. Additional details on the tests used for each specific graph can be found in the figure legends and Supplementary Table 1.

    4. Description of analyses that authors prefer not to carry out

    Reviewer 3

    Major comments:

    Point 5. It is not clear how the constitutive activation of c-Src affects both cell-cell junction and focal adhesion morphology. Did the authors study signaling pathways downstream of c-Src such as the PI3K-AKT pathway?

    c-Src is well known to regulate a multitude of signalling pathways, which was definitively shown in analysis by Ferrando et al. using phosphoproteomics (Ferrando, I.M., et al., Mol Cell Proteomics, 2012. 11(8): p. 355-69.) In this manuscript, our primary emphasis is on elucidating the role of c-Src in governing cell-matrix adhesions and the degradation of the extracellular matrix. We delve into the nuanced connection between focal adhesions (FAs) and VE-cadherin through the actin framework in the discussion (see page 10). Additionally, we highlight that beyond its recognised direct targets in FAs and adherens junctions (AJs), c-Src exerts regulatory influence on these structures through its effects on the actin cytoskeleton.

    The PI3K/AKT pathway is implicated in the progression of vascular malformations in Hereditary Hemorrhagic Telangiectasia (HHT), where patients exhibit rapid vasculature expansion akin to the observed effects upon introducing the c-Src-CA mutation. In HHT, PTEN inhibition triggers heightened activity of VEGFA/VEGFR2 and subsequent AKT kinase activation. Although we have conducted preliminary analysis revealing elevated phospho-AKT, we contend that an in-depth examination of each signaling pathway perturbed downstream of c-Src-CA is beyond the current scope of this manuscript. Our future studies will specifically address this, providing a meticulous exploration of c-Src activity in HHT and its intricate interaction with the AKT pathway.

    Minor comments:

    Point 1: General comment: The authors have predominantly presented composite images with overlapping staining, making it challenging to differentiate between different labels. It would be beneficial if the authors could provide individual channel images along with a merge.

    Given the large numbers of multi-channel composite images, we believe it is not feasible to show each individual channel of every merged image in the manuscript. We have included individual channel images where we believe is appropriate. For example, p-paxillin Y118 (Figure 2), Fibronectin (Figure 4). We are happy to provide individual channel images for any image, where specifically requested, such as in Figure 1E’’ where VE-cadherin channel was added.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, Essebier et al., investigated the impact of constitutive activation of cSrc on endothelial cell behavior during vascular sprouting and homeostasis. The authors generated various mutant versions of cSrc to enable the expression of wild type cSrc, constitutively active cSrc, or cSrc with a dysfunctional kinase domain in HUVEC. They used a range of in vitro methods, including traditional 2D culture techniques and cutting-edge approaches like microfabricated vessels for 3D cell culture. They showed that the constitutive activation of c-Src resulted in a vascular ballooning phenotype both in a 3D angiogenic sprouting assay and in microfabricated blood vessels subjected to shear stress. The expression of this mutant form of c-Src was associated with an increase of focal adhesion size and number and an increase of extracellular matrix degradation. The vascular ballooning phenotype induced by constitutive activation of c-Src was partially rescued by the pharmacological inhibition of the matrix metalloproteinase (MMPs).

    Major:

    • "This was further supported by our observation that there were no changes in proliferation in c-Src mutant cells grown in a 2D monolayer".
      • Figure 1A appears to have increased number of cells in the c-Src-CA condition compared to the control condition. Could the authors quantify the number of cells/area as they did for their 3D vessel model? This would reinforce the idea that the ballooning phenotype they observe is not due to differences in proliferation.
      • Would be strengthened with analysis of another proliferation marker, such as EdU label, which is incorporated only during S phase of the cell cycle. Comparing ki67 staining and EdU staining would provide more insights. Also, using their 3D vessel model for this analysis would increase its relevance.
      • In Figure 1E', cells expressing the constitutively active form of cSrc appear to detach, giving the impression of cell death. Have the authors tested the viability/apoptosis of c-Src-CA cells, particularly in their 3D model?
    • "Therefore, reduction of endothelial cell-cell contacts in c-Src-CA cells may be due to elevated VE-cadherin phosphorylation and subsequent internalisation", "As reduction in cell-cell junction integrity has been shown to increase migratory capacity and sprouting angiogenesis [38], our data suggest that a balanced control of both cell-matrix and cell-cell junctions is essential for mediating migration." In general, it's not clear how constitutively active cSrc affects focal adhesions and cell-cell adhesion and how this is responsible for their ballooning phenotype. The role of the phosphorylation of the VE-Cadherin and cell-cell junctions in this process is not clear either.
      • Further analysis of cell-cell junctions and focal adhesions (co-staining of phosphorylated paxillin and VE-Cadherin) and focal adhesions/fibronectin (like in figure 4C) in the context of cell migration (scratch wound assay) would provide important information to strengthen this notion of balanced control of both cell-matrix and cell-cell junctions.
      • It is not clear how the constitutive activation of c-Src affects both cell-cell junction and focal adhesion morphology. Did the authors study signaling pathways downstream of c-Src such as the PI3K-AKT pathway?
    • "Taken together, these results reveal that proteases produced by c-Src-CA cells are locally secreted at FAs but are membrane bound." The claim that proteases are membrane-bound is not convincingly demonstrated. Could the authors assess whether the constitutive form of cSrc activates the expression of specific genes encoding MMPs by qPCR? Or is it more a matter of the effect of c-Src on the transport of MMPs by microtubules?

    Minor:

    • General comment: The authors have predominantly presented composite images with overlapping staining, making it challenging to differentiate between different labels. It would be beneficial if the authors could provide individual channel images along with a merge.
    • The lab already showed in a previous study that mice lacking c-Src specifically in endothelial cells have reduced blood vessel sprouting, leading to the expectation that the constitutively active form of cSrc would increase sprout number in the sprouting assay. Could the authors explain why the constitutively active form of cSrc induces this vascular ballooning and not an increase in the number of sprouts?
    • In Figure 1A, it would be beneficial to include images from orthogonal views. Indeed, in the c-Src-CA condition, it's not clear whether the vascular ballooning observed represents a cluster of cells or an empty space between the bead and the endothelial cells. (Supp movie 1 helps, but it would be useful to add orthogonal views to the figure)
    • In Figure 1D, the method used to analyze sprout shape is not clear, especially for the c-Src-CA condition where the number of sprouts is close to 0. The figure legend indicates that this measurement corresponds to the shape of the sprouting area. Could the authors clarify and explain their quantification method?
    • "however cells within the vessel still maintained come connections (Fig 1E')": The connections between cells are difficult to see in the images in Figure 1E'. Could the authors provide higher magnification images of the VE-cadherin staining to illustrate these connections between cells?
    • "The reduction in migration correlated with an increase in FA size c-Src-CA expressing cells.": Could the authors give more explanation?
    • Could the authors widen the cell trajectory trace in Supplementary Figure 3A?
    • it is very difficult to distinguish fibronectin fibrils on the images shown in figure 4C. it would be beneficial to change the images.
    • "Treatment of ECs with Marimastat in a fibrin bead sprouting assay resulted in a rescue of the ballooning morphology observed in the c-Src-CA cells" Based on the images displayed in the figure and the associated quantifications, it still appears that c-Src-CA+Marimastat induces a vascular ballooning even if it is less pronounced than in the DMSO condition. Hence, it would be more accurate to describe the observed effect as a "partial rescue". In the microfabricated 3D vessel, in the figure 7A, cell-cell junctions still appear altered by c-Src-CA after the treatment with Marimastat, compared to the c-Src-WT-Marimastat, it would be more appropriate to talk about "partial rescue".
    • In Figure 6A, it seems that there is a decrease in the number of sprouts in the c-Src-DN condition compared to the control condition after the DMSO treatment, which is not observed in Figure 1, could the authors explain why?
    • There is no statistical paragraph in the method section.

    Referees cross-commenting

    Agree that the comments of the reviews all seem reasonable. Since cultured EC do not retain very specialized characteristics, perhaps repeating experiments with many other ECs would not be helpful, but suggest some key experiments be performed with one other type of EC.

    Significance

    General assessment:

    The authors generated different mutant forms of c-Src and used them in innovative 3D endothelial cell culture models. The vascular ballooning phenotype induced by constitutive activation of c-Src is particularly interesting and impressive, especially as it can be reproduced in 2 different culture models. The model of cSrc inducing extracellular matrix degradation specifically at the level of focal adhesions is compelling, although it lacks rigorous support in the 3D model. Further analysis of signaling pathways downstream of c-Src would strengthen the work. The link and the necessity of a balance between cell adhesion and cell-cell junctions are mentioned and have started to be explored, particularly through the phosphorylation of Ve-Cadherin, and more in-depth analysis would strengthen this aspect of the work.

    Advance:

    This study provides new insight on the role of c-Src in vascular homeostasis and during sprouting angiogenesis and starts to explore cross-talk between EC cell junctions and focal adhesions. This study also provides new elements crucial for our understanding of vascular malformations and the implication of cell-adhesion to the extracellular matrix in this process. This study may lead to further investigations into the role of c-Src in tumor angiogenesis.

    Audience:

    Basic research / Specialized

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    Referee #2

    Evidence, reproducibility and clarity

    In this work, Essebier and colleagues have shown that the upregulation of c-Src in endothelial cells results in vascular dilation independently of growth factors or shear stress. The authors have shown that this effect is driven by alteration in the number of focal adhesion and the secretion of matrix metalloproteinases responsible for extracellular matrix remodeling as the inhibition of the MMPs rescues the observed effects.

    This is an elegant work, with well-designed experiments and nice images to illustrate them. Congratulations. Nevertheless, the results not really support the conclusions drawn by the authors. The authors have only used one type of vein endothelial cells from one single donor but they conclude that is effect is general for all endothelial cells. Endothelial cells are very heterogeneous, not only depending on their function and localization, vein, artery or capillary, but also between different organs and in disease (PMID: 22315715, PMID: 28775214, PMID: 31944177, PMID: 33514719).

    The authors, should either repeat some of the key experiments in other type of endothelial cells, maybe arterial or microvasculature cells which are commercially available or at least state that the observations presented in this manuscript apply to HUVECs and discuss whether this would also apply for other cell types. Minor. Although the methods are well written and can be understood. To improve transparency, the authors should reduce the referring to other papers to describe the methods they perform and at least some kind of brief description should be included.

    The authors should report the real p value for their tests. Also when the test is not significant.

    Referees cross-commenting

    I agree with reviewer #1. Description of the statistical methods should be described in the methods. I have nothing else to add to the comments from the other reviewers.

    Significance

    The work presented here by Essebier and colleagues is very well designed and performed. The main strength of the manuscript is the study of the molecular mechanism that regulate the relationship between cells and the extracellular matrix. This is not very well studied in the context of disease. Although all the assays have been performed elegantly, the main limitation of this study is that it has been performed in only one type of endothelial cell. For this reason, it is not possible to extrapolate the conclusions drawn to all endothelial cells like the authors do.

    This work advances our knowledge of endothelial cell biology and it will be of special interest for the vascular biology and development communities.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript demonstrates the effects of overexpression of c-Src variants in HUVEC endothelial cells. The c-Src kinase interacts with cell adhesion machinery, and the manuscript dissects relationships downstream of c-Src with respect to cellular behavior. Transduced wild type, constituent active, dominant negative c-Src is assayed by sprouting in 3D using a bead system, growth of microfabricated vessels under oscillatory flow, focal adhesion analysis, migration analysis, ECM analysis, and by rescue with a matrix Metalloprotease inhibitor.

    Major comments:

    1. p5. Fig 1: The sentence that the dominant negative completely abrogated 'this' phenotype implies that the dominant negative was put into the same cells as the constitutively active mutation. 'Abrogated' means it stops the phenotype, and the phenotype in the sentence prior was constitutively active. It is more accurate to say that the dominant negative was not distinguishable from wild type, which is what the statistics show. No double transfection (DN-CA) was performed.
    2. p.5. Fig 1: the phenotype of the CA cells is fascinating. They expand far beyond their normal territory, but they are held together in a lacy bubble. To me, this looks like a different phenotype from the ballooning that might occur in an arteriovenous malformation in vivo, as in vivo malformations are continuously covered by cells. I understand why the authors might use the term ballooning but given that the cells expand without continuously touching each other, I do not think this is the correct term. Would blebbing, or radial migration in a lace-like discontinuous pattern describe it better?
    3. p. 5. The authors do not describe any relationship to notch signaling. But notch signaling is the mechanism by which a sprout is selected. The CA phenotype shows no selection, and every sprout can continue migration. Did the authors check for any relationship between notch signaling c-Src activation? Does upregulation of C-Src downregulate notch?
    4. The statistical methods are not described in the methods (GraphPad?). These need to be added. Are only significant comparisons plotted? In Fig 6 and 7 only pairwise statistics are shown. If all significant comparisons are plotted, then this means that the comparison between the rescued CA and the treated or untreated control is not significant. This can be thought of as a partial rescue towards a wild type, but it is definitely not a full rescue. None of the statistical comparisons in Figure 6 or 7 show significant comparisons to wildtype. This needs more discussion.
    5. Mmp activity is inferred, but not measured. This is a limitaion as the assumption is that marimostat acting through the expected pathway.

    Minor concerns:

    1. Fig 5D. The presentation of the data in this graph is difficult to understand. It is trying to show the proportion of mScarlet in sprouts or balloons a percentage of all the scarlet cells. It would be better to have all cells represented in one bar, distributed between sprout and balloon in that one bar. i.e., for the control and dominant negative, the bars would be all black and then for the CA it would be all white. The zero data points are confusing. A proportions graph should be investigated here.
    2. The methods for vessel coverage for quantification in figs 1 and 7 are missing.

    Referees cross-commenting

    The comments from the other reviewers seem reasonable.

    Significance

    The work is well executed and takes a mechanistic approach. The images are well put together and the movies significantly add to the manuscript. The phenotype describes highly unusual endothelial behavior, which is of interest, and an advance in the field for its novelty. Linking cSrc to downstream signalling including mmps and demonstrating a rescue is also novel and a strength. This is a conceptual advance in the relationship between a kinase and cell behaviour in 3D.

    Understanding this mechanism may be useful in understanding enlarged vessels in vascular malformations, although the direct relevance is not clear due to limitations of using cultured cells in artificial environments, lacking, for instance, support by secondary cells and ECM that might be contributed by support cells and perhaps modulate the phenotype.

    The audience would be specialized in the basic research community.

  9. Version published to 10.1101/2023.09.15.557868v1 on bioRxiv