Peer review of: Endothelial cell clonal expansion in the development of cerebral cavernous malformations (2019)

Reviewers' comments:  

 

Reviewer #1 (Remarks to the Author):  

 

Cerebral cavernous malformation is a severe neurovascular familial or sporadic disease that is 
characterised by capillary  

venous cavernomas,and is due to loss of function mutations to one  

of three CCM genes. The authors previously noted that in sporadic cavernomas only a small fraction 
of endothelial cells shows mutated CCM genes and that in a mouse model of the human disease, 
endothelial cells lining the lesions have different transcriptome features from much of the 
surrounding endothelium.  

In this report, the authors state that cavernomas originate from clonal expansion of a small number 
of Ccm3-null endothelial cells  

that express both endothelial and mesenchymal/stem cell markers. Second, this small population of 
mutant cells is purported to attract surrounding wild-type cells to induce them to express  

mesenchymal/stem-cell markers. The authors conclude these characteristics of Ccm3-null cells are 
reminiscent of "tumour initiating cells' that are responsible for tumour growth and metastatic 
differentiation and that CCM has benign tumour characteristics. These are the main claims.  

 

There are several points that need to be addressed to solidify the claims:  

1) How do the authors define a clone in the different tissues and at different ages of the mice? This 
is a key point. Is the definition based on a certain number of contiguously expressing cells within a 
vessel: how many cells comprise a clone (line 90-95; 123-130?  

2) While some evidence for the ability of the mutant cells to modify the behavior of the wild-type 
cells in vitro, it was not proven that the mutant cells possess the capacity to recruit and cause the 
formation of CCM in vivo. This could be readily accomplished using simple Matrigel plug assays 
containing the mutant cells (labled) implanted in the Cdh5-reporter mice to distinguish the mutant 
from wild type cells. Generation of cavernous hemangiomas in the plugs would prove the mutant 
cells were indeed capable of recruiting and causing these lesions (without the recruited cells 
becoming genetically modified).  

3) (lines 165-175) Do the recruited wild-type cells lose their endothelial functional capacities (vessel 
forming potential) when they upregulate the EMT profile of genes? If the cells that upregulate the 
EMT genes are simply left in culture with removel of the mutant genes, do the wild-type cells revert 
back to a normal endothelial phenotype and function?  

 

 

Reviewer #3 (Remarks to the Author):  

 

Malinverno et al describe clonal endothelial cells (ECs) that contribute to the pathobiology of 
Cerebral Cavernous Malformations (CCM). The study is a followup from several recent reports from 
the Dejana lab describing ECs with mesenchymal-like features in CCM. The authors use an elegant 
reporter system (R26R-Confetti mice) in mice with conditional CCM3 deletion in ECs to demonstrate 
clonal expansion of ECs in early lesions which eventually become “mixed” due to recruitment of ECs 
that are CCM3+/+. The authors suggest that CCM3 null ECs recruit CCM wildtype ECs into CCM 
lesions via BMP6. They also suggest that CCM mutations within a small group of EC progenitors are 
primarily responsible for driving the formation of CCM lesions although this data needs further 
elaboration. Overall, the study presents a compelling model for a “field effect” in CCM whereby 
CCM3-/- ECs can alter the behavior of CCM3+/+ ECs nearby thereby contributing to CCM 
progression.  

 

Specific critique (in no particular order)  

 

The authors should reconsider the use of the word “transform” as in “…CCM3-/- ECs transform 
CCM3+/+ ECs…”. “Transform” generally refers to mutations that enable the acquisition of malignant 
traits and the authors do not demonstrate that the cells are indeed “transformed ” in this way. A 
different term might be chosen to make this distinction.  

 

With regards to clonal expansion of CCM3-/- ECs: the evidence for this is based on color labeling in 
the confetti background; however, because labeling is stochastic, it is theoretically possible that two 
neighboring cells are labeled with the same color fluorophore but are not actually derived from the 
same parent cell. The authors should in the least discuss this possibility or provide additional 
experimental evidence that the ECs in CCM lesions are indeed clonal.  

 

It is not clear why the particular genes assessed (Klf4, Id1, pSMAd3, Sca1, etc) are called EndMT 
markers - what distinguishes these genes as EndMT markers? Aren’t these genes expressed by 
normal ECs at some level even basally? The immunofluorescence staining in fig 3e-h might be more 
convincing with normal vessels as a comparison and help indicate whether these markers typify 
EndMT or are enriched in areas where EndMT is or has occurred.  

 

Rescue experiments should be carried out using CCM3 re-expression in CCM3-/- ECs. Does this 
suppress EndMT marker genes or correct their (mal)functional properties?  

 

What is the fate/function of CCM3-/- ECs in vivo? The authors should re-inject these cells into 
syngeneic mice to assay their morphology and function (e.g. anastamosis/perfusion). Do they form 
aberrant vascular structures when re-injected? These experiments could be done using CCM3 rescue 
in CCM3-/- ECs (see above).  

 

Since the authors suggest that CCM3-/- ECs expand clonally in the CCM lesions, is it assumed that 
this expansion is via proliferation? If so, what is the link between CCM3 and cell cycle? Is deletion of 
CCM3 sufficient to drive S-phase or inhibit apoptosis in ECs? The authors should test this possibility 
experimentally.  

 

The endothelial progenitor marker staining (CD157/CD200) is difficult to see and needs 
quantification. This staining also looks perivascular? Related to this, what fraction of Procr+ cells are 
labelled? Staining for Procr should be carried out. The concept that a small number of EC progenitors 
are the “drivers” of CCM lesions is compelling, but the data needs to be presented more clearly.  

 

Can the authors assay the CCM gene product (PDCD10) and show mosaicism in human CCM lesions 
(PDCD10+ ECs and PDCD10- ECs)?  

 

Do culture expanded CCM3-/- ECs retain or show enrichment for EC progenitor markers compared 
to their CCM3+/+ counterparts?  

 

The authors suggest that CCM3-/- ECs recruit/convert CCM3+/+ ECs, but do CCM3+/+ ECs 
block/revert/normalize CCM3-/- ECs in any way? This should be tested experimentally.  

 

Is there any evidence that CCM3+/+ ECs are recruited systemically (i.e. bone marrow)? Or are these 
locally-derived ECs from nearly vessels? A bone marrow origin is doubtful but maybe a point worthy 
of discussion if the authors have an opinion or data.  

 

 

Reviewer #4 (Remarks to the Author):  

 

The work by Malinverno and colleagues builds upon previous findings by the Dejana laboratory 
suggesting that endothelial cells (ECs) lining cavernomas, the hallmark vascular lesions of the 
cerebrovascular disorder Cerebral Cavernous Malformations (CCM), are morphologically and 
molecularly different from ECs of the same vessel outside the lesion site. Familial forms of CCM are 
underlain by mutations in one of three genes (CCM1-3), and the two-hit mechanism (a germline 
mutation followed by a somatic mutation in a small number of ECs) has been employed to explain 
disease pathogenesis. In this manuscript, the authors test the hypothesis that CCM lesions originate 
from a small number of Ccm3-mutant ECs, which expand clonally, recruit surrounding wild type ECs, 
and induce their molecular and morphological transformation.  

 

The manuscript addresses an interesting and novel idea, which is tested using acute and chronic 
mouse models of CCM3 disease, additional inducible transgenic mice, as well as in vitro co-culture 
assays of wild type or Ccm3 mutant ECs. The authors present a wealth of diverse and detailed data 
arguing in support of the proposed hypothesis. The study has considerable strengths, but also 
significant weaknesses. The major strength of the study is the novelty of the idea tested, namely, 
that clonal expansion of ECs mechanistically explains cavernoma formation. The major weakness, on 
the other hand, is that the conclusion that cerebral cavernomas arise from clonal expansion of ECs 
(as also stated in the title) is not fully supported by the data.  

 

Below are the major points I wish to raise about the study:  

 

1) Figure 1  

 

The authors should include a picture of wild type retina, and not of (or, in addition to) wild type 
brain (current panel 1a). If the goal is to illustrate the clonal nature of retinal lesions, then the 
reference should be an image of the wild type retina. Is it possible that cavernomas have clonal 
origin because the arise from a vessel that is itself clonal?  

 

The lesions (magnification panel 1) in panels 1c and 1c’ are shown as being representative of a 
“larger” lesion; however, they do not have the appearance of mature lesions with multiple caverns. 
Moreover, a section through a (any) lesion is not necessarily perpendicular to the wall of the dilated 
vessel (as it could be at an angle). Consequently, without a true 3D reconstruction, (and not just 
assessment of 10 thin sections [unclear if they are serial]), lesion area and diameter cannot be 
correctly assessed. This is an important point for the premise of the study, as small diameter lesions 
are said to be clonal, whereas, in contrast, large cavernomas to be mosaic. This point becomes even 
more relevant as it is difficult to assess the non-clonality of the large lesions presented in panel 1d 
(too few cells of a different origin compared to the vast majority of the lesion that appears to be 
rather clonal).  

 

2) Extended Figure 1a  

 

Please correct the schematic (it is indicated lethal at P10, however the text -line 98- states “lethal in 
around 12 days”  

 

3) Figure 2d, e and Extended Figure 1d (text line 104)  

 

The comparison attempted here is not valid – the acute model (P8) cannot be compared to the 
chronic model at P30. The comparison should be carried out within the same model.  

 

4) In order to conclude that cavernomas form via progression from a clonal, small/immature/early 
lesion to a non-clonal, larger/mature lesion (in which wild type cells are being recruited and induced 
to change in the presence of Ccm3-mutant cells), lesion progression (and accompanying molecular 
changes) should be documented in the chronic models. Specifically, staining with EMT markers over 
time will be necessary on smaller lesions that are 100% positive vs. larger (more mature) lesions, 
which should be mosaic. The authors performed a similar comparative analysis focusing on KLF4-
positive cells (Fig. 2e), which should be extended to earlier time points in the same (chronic) model.  

 

5) In Figure 2f-m (presumably corresponding to the slow progression/chronic model – please clarify 
in the legend), the quality of the immunofluorescence could be improved. PECAM1 staining appears 
not to outline the lining of the lesions in its entirety; as a consequence, some, presumably, ECs 
appear positive for the mesenchymal markers, but lack PECAM1 staining. Can the authors show in 
the same model that smaller/immature/clonal lesions are 100% positive for EMT markers, whereas 
larger/mature lesions show a significant reduction of ECs that are positive for EMT markers?  

 

6) Extended Figure 2c  

 

The in situ hybridization data are not convincing: this figure could be improved by DIC microscopy to 
visualize the outline of the cells; please comment on the staining outside the lesions.  

 

7) Extended Figure 3  

 

Please specify that these are immortalized lung endothelial cells  

 

8) Figure 4  

 

i) Panels b, d, and e: it is very difficult to assess the number of wild type ECs recruited in the Ccm3-
mutant spheroids.  

 

ii) Panel f and lines 174 and 189. The observed changes in migration rate in response to conditioned 
media from Ccm3-mutant EC cultures are mild and do not support the conclusion of the involvement 
of secreted factors (see also points iii and iv below).  

 

iii) Panel f: There is a discrepancy between the data on the rate of migration described in this panel 
(3 um/hr with conditioned media from wild type cultures vs. 8 um/hr with conditioned media from 
Ccm3-mutant cultures) and data in Extended Figure 7e (7 um/hr with vehicle as negative control). 
How do the authors explain this?  

 

iv) Line 162 and Extended Figure 6. As the authors mention, involvement of cell-cell contacts in 
mediating the recruitment of wild type cells by Ccm3-mutant ECs is indeed plausible.  

v) Panel g (and lines 165-168): the comparison here is between wild type cells isolated from the 
spheroids vs. wild type cells cultured as a monolayer. This is problematic, and a more accurate 
comparison would have been with wild type cells that have not been recruited/encapsulated in the 
spheroids, yet cultured in the same dish. An additional problem is that by definition cells that grow 
in 3D (such as within a spheroid), undergo morphological (and molecular) changes that distinguish 
them from the same cells grown as a monolayer.  

 

9) line 170: how do the authors explain the increase on Fsp1 relative expression only in the presence 
of BMP6, but not upon exposure to conditioned media from Ccm3-mutant cultures?  

 

10) Although the source of the different materials used in every figure/panel is described in 
Methods, having to constantly refer to the Methods to retrieve this information is disruptive. It 
would be preferable if this information is also provided as needed throughout the text and in the 
figure legends (e.g. ECs in culture are either primary brains ECs or lung ECs; FACS is performed not 
on mice, but specifically on brains, etc.)  

 

In summary, the work presented in this manuscript is intriguing and proposes a novel mechanism to 
explain the formation of CCM lesions. However, additional experiments are needed to support the 
conclusions reached by the authors, as well as the definitive nature of the title-statement, which in 
this reviewer’s opinion is very strongly worded, without being fully substantiated by the findings as 
these are currently detailed, and thus should be revised.