The myeloid cell-driven transdifferentiation of endothelial cells into pericytes promotes the restoration of BBB function and brain self-repair after stroke

Reviewer #1 (Public review):

Summary:

Using lineage tracing and single-cell RNA sequencing, Li et al. reported brain ECs can differentiate into pericytes after stroke. This finding is novel and important to the field.

Strengths:

Detailed characterization of each time point and genetic manipulation of genes for study role of ECs and E-pericyte.

Weaknesses:

Genetic evidence for lineage tracing of ECs and E-pericytes requires more convincing data that includse staining, FACS, and scRNA-seq analysis.

Reviewer #2 (Public review):

Summary:

In this manuscript, Li and colleagues study the fate of endothelial cells in a mouse model of ischemic stroke. Using genetic lineage tracing approaches, they found that endothelial cells give rise to non-endothelial cells, which they term "E-pericytes." They further show that depleting these cells exacerbates blood-brain barrier leakage and worsens functional recovery. The authors also provide evidence that endothelial-to-mesenchymal transition, myeloid cell-derived TGFβ1, and endothelial TGFβRII are involved in this process. These are potentially interesting findings, however, the experimental evidence that endothelial cells undergo transdifferentiation to non-endothelial cells is weak, as is the evidence that these cells are pericytes. Addressing this foundational weakness will facilitate the interpretation of the other findings.

Strengths:

(1) The authors address an important question about blood vessel function and plasticity in the context of stroke.

(2) The authors use a variety of genetic approaches to understand cell fate in the context of stroke. Particularly commendable is the use of several complementary lineage tracing strategies, including an intersectional strategy requiring both endothelial Cre activity and subsequent mural cell NG2 promoter activity.

(3) The authors address upstream cellular and molecular mechanisms, including roles for myeloid-derived TGFβ.

Weaknesses:

(1) The authors use Cdh5-CreERT2; Ai47 mice to permanently label endothelial cells and their progeny with eGFP. They then isolate eGFP+ cells from control and MCAO RP7D and RP34D brains, and use single-cell RNA-seq to identify the resulting cell types. Theoretically, all eGFP+ cells should be endothelial cells or their progeny. This is a very powerful and well-conceived experiment. The authors use the presence of a pericyte cluster as evidence that endothelial-to-pericyte transdifferentiation occurs. However, pericytes are also present in the scRNA-seq data from sham mice, as are several other cell types such as fibroblasts and microglia. This suggests that pericytes and these other cell types might have been co-purified (e.g., as doublets) with eGFP+ endothelial cells during FACS and may not themselves be eGFP+. Pericyte-endothelial doublets are common in scRNA-seq given that these cell types are closely and tightly associated. Additionally, tight association (e.g., via peg-socket junctions) can cause fragments of endothelial cells to be retained on pericytes (and vice-versa) during dissociation. Finally, it is possible that after stroke or during the dissociation process, endothelial cells lyse and release eGFP that could be taken up by other cell types. All of these scenarios could lead to the purification of cells that were not derived (transdifferentiated) from endothelial cells. The authors note that the proportion of pericytes increased in the stroke groups, but it does not appear this experiment was replicated and thus this conclusion is not supported by statistical analysis. The results of pseudotime and trajectory analyses rely on the foundation that the pericytes in this dataset are endothelial-derived, which, as discussed above, has not been rigorously demonstrated.

(2) I have the same concern regarding the inadvertent purification of cells that were not derived from endothelial cells in the context of the bulk RNA-seq experiment (Figure S4), especially given the sample-to-sample variability in gene expression in the RP34D, eGFP+ non-ECs-group (e.g., only 2/5 samples are enriched for mesenchymal transcription factor Tbx18, only 1/5 samples are enriched for mural cell TF Heyl). If the sorted eGFP+ non-ECs were pericytes, I would expect a strong and consistent pericyte-like gene expression profile.

(3) The authors use immunohistochemistry to understand localization, morphology, and marker expression of eGFP+ cells in situ. The representative "E-pericytes" shown in Figure 3A-D are not associated with blood vessels, and the authors' quantification also shows that the majority of such cells are not vessel-associated ("avascular"). By definition, pericytes are a component of blood vessels and are embedded within the vascular basement membrane. Thus, concluding that these cells are pericytes ("E-pericytes") may be erroneous.

(4) CD13 flow cytometry and immunohistochemistry are used extensively to identify pericytes. In the context of several complementary lineage tracing strategies noted in Strength #2, CD13 immunohistochemistry is the only marker used to identify putative pericytes (Figure S3J-M). In stroke, CD13 is not specific to pericytes; dendritic cells and other monocyte-derived cells express CD13 (Anpep) in mouse brain after stroke (PMID: 38177281, https://anratherlab.shinyapps.io/strokevis/).

(5) The authors conclude that "EC-specific overexpression of the Tgfbr2 protein by a virus (Tgfbr2) decreases Evans blue leakage, promotes CBF recovery, alleviates neurological deficits and facilitates spontaneous behavioral recovery after stroke by increasing the number of E-pericytes." All data in Figure 10, however, compare endothelial Tgfbr2 overexpression to a DsRed overexpression control. There is no group in which Tgfbr2 is overexpressed but "E-pericytes" are eliminated with DTA (this is done in Figure 9B, but this experiment lacks the Tgfbr2 overexpression-only control). Thus, the observed functional outcomes cannot be ascribed to "E-pericytes"; it remains possible that endothelial Tgfbr2 overexpression affects EB leakage, CBF, and behavior through alternative mechanisms.

(6) Single-cell and bulk RNA-seq data are not available in a public repository (such as GEO). Depositing these data would facilitate their independent reevaluation and reuse.

Reviewer #3 (Public review):

Summary:

The data and experiments presented in that study convincingly show that a subpopulation of endothelial cells undergo transformation into pericyte-like cells after stroke in mice. These so-called "E-pericytes" are protective and might present a new target for stroke recovery. The authors used a huge battery of different techniques and modified signaling pathways and cellular interactions using several genetic and pharmacological tools to show that TGFbeta and EndoMT are causes of this transformation.

Strengths:

The amount of different genetic and pharmacological approaches in combination with sophisticated techniques such as single-cell RNAseq is impressive and convincing. The results support their conclusions and the authors achieved their aims. The findings will strongly impact the field of cerebrovascular recovery after stroke and might open up new therapeutic targets.

Weaknesses:

The written and graphic presentation of the findings needs substantial improvement. Language editing is strongly recommended (there are a lot of spelling and grammatical errors in the text and illustrations, including legends).