Pial collaterals develop through mosaic colonization of capillaries by arterial and microvascular endothelial cells

Reviewer #1 (Public Review):

The paper by Perovic and colleagues describes how important blood vessels called collaterals form during development and remodel/expand upon injury to the brain. These vessels are conduits between arteries that do not have strong blood flow physiologically but upon injury can compensate for conduit loss. Published work by others is largely descriptive and does not address the cellular sources of collaterals over time. Here elegant lineage tracing is used to better understand the source of vascular endothelial cells during embryonic development, and how these lineages contribute to remodeling upon injury. The work is ambitious and important as collateral capacity can strongly influence the trajectory of outcomes with vascular blockage. The work reveals that proliferative arterial EC is the primary contributor to the collaterals developmentally, with a small contribution from capillary/venous EC, and that this shifts to almost completely arterial contribution from birth onward. There are several aspects of the work that, if addressed, would strengthen the study and better support the interesting and novel conclusions, including analysis of non-collateral lineage contributions, more careful interpretation of fixed image data, and more careful annotation of the image panels.

Reviewer #2 (Public Review):

Pial collateral vessels are anastomotic connections that cross-connect distal arterioles of the middle, anterior, and posterior cerebral arteries. With respect to ischemic stroke, good pial collateral flow positively correlates with decreased infarct volume and improved recovery; accordingly, optimizing collateral flow represents an important intervention for limiting stroke damage. The goal of this study was to determine the endothelial cell (EC) subtype(s) that contribute to the embryonic and neonatal development of pial collaterals and their expansion in response to stroke. To this end, the authors used lineage tracing methods in the mouse, labeling arterial endothelial cells (using Bmx-CreERT on switch line, R26mTmG) or venous and microvascular endothelial cells (using Vegfr3-CreERT on R26mTmG) and assessing pial collaterals via confocal microscopy. The authors convincingly demonstrate that arterial-lineage ECs comprise the majority of pial collateral ECs during development and in adulthood, with a minor contribution from pial plexus-derived microvascular ECs that decline over time. They also convincingly demonstrate that pial collateral outward remodeling after experimentally-induced stroke (distal middle cerebral artery occlusion, or dMCAO) involves, at least in part, local proliferation of arterial-lineage ECs. The latter is intriguing given that arterial ECs generally leave the cell cycle. While these conclusions are quite solid, some key details are missing that could improve analysis, and some important caveats are not addressed. Moreover, less convincing are mechanistic claims that pial collaterals form via a migratory process of "mosaic colonization" of a preexisting vessel.

1. It is difficult to understand whether individual collaterals are truly mosaic vessels, or whether arterial or venous/microvascular lineage ECs predominate in any particular region of the pial collateral vasculature. This is due to a number of methodological reasons: arterial and venous/microvascular contributions to pial collaterals were assessed independently, only a few (and in some cases, just one) collaterals were analyzed in each mouse, and regionality/location of collaterals was not addressed. Additionally, the inefficiency and variability of EC labeling, especially with the Vegfr3-CreERT line (Fig. S1, ~6-30%), compounds this problem.

2. The identification of "pre-collateral" vessels requires further support. The authors define these vessels by their connection to the feeding artery, their (often) larger diameter, and their more pronounced ICAM2 expression. While most of these criteria are demonstrated in Figure S3, it is not apparent how these vessels were defined in Figure 4, which lacks specific annotation of each of these identifying criteria. As the identification of these novel vessels is one of the key findings of this paper, a more robust method of unambiguously defining them is warranted.

3. The conclusion that collateral-forming ECs migrate in the direction of flow into preexisting vessels is not well supported. The authors state that the presence of filopodial projections (Figure 4) supports this conclusion. However, filopodia number and directional polarization/orientation were not quantified, and "intercalation movements"/migration, per se, cannot be inferred from these static images.

4. In Figure 5, the simplest explanation for relative Cx40 expression in different vessels is the absence (low expression) or presence (high expression) of flow. This figure provides little mechanistic insight beyond this already-known relationship, and it is unclear how many times this experiment was performed (there is no N, no quantification or correlation).

5. There is no statistical analysis in this work. This is justified by the authors by their admission that the study is of a "descriptive nature and...exploratory design."

Reviewer #3 (Public Review):

Summary:
These studies focus on a very interesting, understudied phenomenon in vascular development - the formation of pial collaterals between cerebral arteries. Understanding the mechanism(s) that regulates this process during normal development could provide important insights for the treatment of adult stroke patients, for which repair is highly dependent on collateral formation. Insights may also be relevant to other collateral-dependent diseases, such as heart disease and chronic peripheral ischemia.

Strengths:
The investigators use lineage tracing and 3D imaging to show that, in mouse embryos, endothelial cells (ECs) predominantly from Bmx+ arteries and some from the Vegfr3+ microvasculature, invade pre-existing pre-collateral vascular structures in a process they termed "mosaic colonization", and arterialization of the vessel segments is said to occur concurrently with colonization, although details about EC phenotypes are lacking. Growth of the collaterals in response to ischemic injury relies on local replication of the ECs within the collaterals and not further recruitment from veins and the microvasculature. Although detailed molecular mechanisms are not provided, demonstration of the "cellular mechanism" of pial collateral vascularization is novel.

Weaknesses:
Nonetheless, there are some issues that should be addressed, particularly to clarify the phenotype of the ECs forming the collaterals and expanding in response to injury; only their "origin" was traced and not their identity/growth after labeling in Bmx+ vessels.

Author Response

Public Reviews:

Reviewer #1 (Public Review):

The paper by Perovic and colleagues describes how important blood vessels called collaterals form during development and remodel/expand upon injury to the brain. These vessels are conduits between arteries that do not have strong blood flow physiologically but upon injury can compensate for conduit loss. Published work by others is largely descriptive and does not address the cellular sources of collaterals over time. Here elegant lineage tracing is used to better understand the source of vascular endothelial cells during embryonic development, and how these lineages contribute to remodeling upon injury. The work is ambitious and important as collateral capacity can strongly influence the trajectory of outcomes with vascular blockage. The work reveals that proliferative arterial EC is the primary contributor to the collaterals developmentally, with a small contribution from capillary/venous EC, and that this shifts to almost completely arterial contribution from birth onward. There are several aspects of the work that, if addressed, would strengthen the study and better support the interesting and novel conclusions, including analysis of non-collateral lineage contributions, more careful interpretation of fixed image data, and more careful annotation of the image panels.

We thank the reviewer for appreciating the ambition, importance and novelty of our work, and for the constructive suggestions for improvements.

Reviewer #2 (Public Review):

Pial collateral vessels are anastomotic connections that cross-connect distal arterioles of the middle, anterior, and posterior cerebral arteries. With respect to ischemic stroke, good pial collateral flow positively correlates with decreased infarct volume and improved recovery; accordingly, optimizing collateral flow represents an important intervention for limiting stroke damage. The goal of this study was to determine the endothelial cell (EC) subtype(s) that contribute to the embryonic and neonatal development of pial collaterals and their expansion in response to stroke. To this end, the authors used lineage tracing methods in the mouse, labeling arterial endothelial cells (using Bmx-CreERT on switch line, R26mTmG) or venous and microvascular endothelial cells (using Vegfr3-CreERT on R26mTmG) and assessing pial collaterals via confocal microscopy. The authors convincingly demonstrate that arterial-lineage ECs comprise the majority of pial collateral ECs during development and in adulthood, with a minor contribution from pial plexus-derived microvascular ECs that decline over time. They also convincingly demonstrate that pial collateral outward remodeling after experimentally-induced stroke (distal middle cerebral artery occlusion, or dMCAO) involves, at least in part, local proliferation of arterial-lineage ECs. The latter is intriguing given that arterial ECs generally leave the cell cycle. While these conclusions are quite solid, some key details are missing that could improve analysis, and some important caveats are not addressed. Moreover, less convincing are mechanistic claims that pial collaterals form via a migratory process of "mosaic colonization" of a preexisting vessel.

We thank the reviewer for the careful assessment and suggestions for improvements. Claiming migratory behaviour from static images is indeed always tricky and comes with caveats. Our conclusions however are based on the appearance of cells in locations where they are not found at earlier stages. Given that we could exclude persistent recombination, a sound conclusion must be that cells appear in the new location through some means of translocation. Given our experience with the morphology of migrating cells in vivo, the appearance of polarized filopodial structures coinciding with the direction of observed appearance of cells at progressive later stages, strongly suggests active migration. Moreover, these highly migrating cells also exhibit ICAM2 positivity, suggesting that they are directly lining the pre-collateral lumen. In our explanation of how the immigration might occur, we would need to consider solitary cell migration through interstitial space, or rather intercalation movement. The active participation of migrating cells in lumen formation of the nascent pre-collateral suggests intercalation, but further analysis needs to be performed (such as a detailed analysis of cell-cell junctions or sustained apico-basal polarity). The conclusion that such a process highlights mosaic colonization of preexisting vessels is tightly linked to the demonstration of continuous lumen, whilst being found in a vessel without lineage marker, but beginning expression of arterial markers such as Cx40.

1. It is difficult to understand whether individual collaterals are truly mosaic vessels, or whether arterial or venous/microvascular lineage ECs predominate in any particular region of the pial collateral vasculature. This is due to a number of methodological reasons: arterial and venous/microvascular contributions to pial collaterals were assessed independently, only a few (and in some cases, just one) collaterals were analyzed in each mouse, and regionality/location of collaterals was not addressed. Additionally, the inefficiency and variability of EC labeling, especially with the Vegfr3-CreERT line (Fig. S1, ~6-30%), compounds this problem.

Factual error: 6 - 22% (not 30)

The reviewer is correct in their statement that the independent assessment of contribution makes it difficult to locally demonstrate mosaicism. However, we are not aware of a method that could trace two different populations from different sources using recombination genetics simultaneously. Mosaicism however can be concluded from two observations independently. One, we find contribution from an alternative source that at the time point of labelling does not colocalize with arterial BMX lineage cells. Second, the BMX-lineage labelling is never complete in the collaterals, at least at developmental stages. Future work using scRNA seq may shed more light onto the degree of mosaicism. However at this point, the data strongly suggest mosaicism, even if the majority of the cells are of the BMX-lineage. The comment on inefficiency or variability of labelling in particular with the Vegfr3-CreERT line is interesting. At this point, we cannot rule out that the observed variability is due to intrinsic variability in expression, rather than inefficient recombination, or variability thereof. With our current tools we cannot easily distinguish between the two. Again, we hope that future studies with scRNA seq will be able to shed more light onto this interesting biology. Finally, we have not carefully assessed regionality, but have not seen obvious correlations with the degree of mosaicism. It is however important to note that in no case did we just examine one collateral per hemisphere. Each data point is an average of all collaterals from a part of a given collateral zone (imaging region). Usually, it is possible to image 2-4 collateral regions in each embryo. We always imaged multiple collaterals per animal, but sometimes only one region was imaged (due to technical issues).

1. The identification of "pre-collateral" vessels requires further support. The authors define these vessels by their connection to the feeding artery, their (often) larger diameter, and their more pronounced ICAM2 expression. While most of these criteria are demonstrated in Figure S3, it is not apparent how these vessels were defined in Figure 4, which lacks specific annotation of each of these identifying criteria. As the identification of these novel vessels is one of the key findings of this paper, a more robust method of unambiguously defining them is warranted.

We agree that it would be fabulous to have a unique marker at hand that identifies pre-collaterals. Our careful analysis of the distribution of the markers we tested, firmly established that the levels of ICAM2 expression nicely highlight structures that become colonized by these BMX lineage cells. Cx40 staining also confirmed this impression. We will attempt better annotation based on these markers to help the reader appreciate these findings. The combination of anatomical location and connection pattern with the stronger ICAM2 staining in our hands is a highly reliable and unambiguous identifier of what we called “pre-collaterals”.

1. The conclusion that collateral-forming ECs migrate in the direction of flow into preexisting vessels is not well supported. The authors state that the presence of filopodial projections (Figure 4) supports this conclusion. However, filopodia number and directional polarization/orientation were not quantified, and "intercalation movements"/migration, per se, cannot be inferred from these static images.

The reviewer is correct that claiming migration from static images is always difficult. As stated above, we base our conclusions on the progressive appearance of cells exhibiting migratory behavior, as well as the morphology including filopodia. Although we indeed didn’t quantify filopodia, these structures are in our experience not found on endothelial cells that do not engage in migration. Their consistent presence, and directionality is strongly suggestive of movement. . We will attempt to clarify this better in the text and the figures.

1. In Figure 5, the simplest explanation for relative Cx40 expression in different vessels is the absence (low expression) or presence (high expression) of flow. This figure provides little mechanistic insight beyond this already-known relationship, and it is unclear how many times this experiment was performed (there is no N, no quantification or correlation).

Flow is indeed one component of what regulated Cx40. However, a key point of this figure is to show that Cx40 expression can precede the recruitment of BMX lineage cells. This is important to distinguish whether arterial identity is only achieved by recruitment of BMX lineage cells, or exists in certain vessels (for example because they may have more flow) already before this colonization event. It suggests that the BMX population may rather serve to consolidate arterial state, as other structures that may have been Cx40 before, but do not become colonized lose arterial identity? We disagree that this finding does not contribute important information. If only BMX-lineage cells would express Cx40, the conclusion would be very different. This is not a question of how much, but of whether arterialization requires the recruitment of particular cells, or is induced in vessels that adopt arterial identity. This is not a singular observation and we will add the N number onto the figure legend.

1. There is no statistical analysis in this work. This is justified by the authors by their admission that the study is of a "descriptive nature and...exploratory design."

This is correct.

Reviewer #3 (Public Review):

Summary:

These studies focus on a very interesting, understudied phenomenon in vascular development - the formation of pial collaterals between cerebral arteries. Understanding the mechanism(s) that regulates this process during normal development could provide important insights for the treatment of adult stroke patients, for which repair is highly dependent on collateral formation. Insights may also be relevant to other collateral-dependent diseases, such as heart disease and chronic peripheral ischemia.

Strengths:

The investigators use lineage tracing and 3D imaging to show that, in mouse embryos, endothelial cells (ECs) predominantly from Bmx+ arteries and some from the Vegfr3+ microvasculature, invade pre-existing pre-collateral vascular structures in a process they termed "mosaic colonization", and arterialization of the vessel segments is said to occur concurrently with colonization, although details about EC phenotypes are lacking. Growth of the collaterals in response to ischemic injury relies on local replication of the ECs within the collaterals and not further recruitment from veins and the microvasculature. Although detailed molecular mechanisms are not provided, demonstration of the "cellular mechanism" of pial collateral vascularization is novel.

Weaknesses:

Nonetheless, there are some issues that should be addressed, particularly to clarify the phenotype of the ECs forming the collaterals and expanding in response to injury; only their "origin" was traced and not their identity/growth after labeling in Bmx+ vessels.

We thank the reviewer for pointing out the importance and novelty of our findings, and for the constructive suggestions for improvements. We indeed focussed here on origin and an attempt to distinguish how the cells arrive in their location rather than on their phenotype. We have performed detailed phenotypic analysis including EM analysis of collaterals but without the ability to connect these to the traced lineages. We therefore chose to leave these data for a separate manuscript. Future work will attempt to fully characterize these populations including their transcriptome using scRNA seq. However, isolating collateral ECs to faithfully characterize them is very challenging, and will not be a part of this manuscript. We have performed stainings for various arterial markers, with variable success.. Nevertheless, a full functional study will be part of future work.