Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes

Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFβ signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes.

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Cell line source(s) Mouse primary brain pericytes were isolated according to previously published protocols. A detailed description of the procedure is available in the Methods section (Primary brain pericytes isolation and culture).

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Expression of known pericyte markers was assessed by RT-qPCR and immunohistochemistry.

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Primary brain pericytes were not tested for mycoplasma contamination.

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In case of peakranger we called 51774 and 67920 peaks respectively with FDR<0.05 and 5-fold enrichment of IP over input reads.
In case of macs2 we called 27737 and 27649 peaks respectively with FDR<0.05 and 3-fold enrichment of IP over input reads.
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2% FCS) was added and the tissue homogenate was mixed with 1.7 volumes of 22% albumin fraction V (BSA, Carl Roth, 8076.2). After centrifugation (1000 g, 12 min at RT) the supernatant was removed and the cell pellet resuspended in 5 mL of FACS buffer, filtered through a 40 μm nylon mesh (Corning, 352340) and centrifuged (300 g, 5 min at RT). The cell pellet, consisting mostly of single cells was resuspended in a suitable volume of FACS buffer together with the antibodies used for staining which are described in detail in the Methods section (Fluorescence Assisted Cell Sorting (FACS) of brain pericytes and endothelial cells).

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Cell sorting was performed in a FACS Aria IIu (BD Biosciences) with a 70 μm nozzle.
Cell population abundance Purity of sorted fractions is assessed by gene expression analysis of putative markers for the specific cell population with respect to the input (single cell suspension before sorting) and the other cell fraction sorted (i.e. pericytes vs. endothelial cells).

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Exclusion of debris and doublets together with single cells selection was based on forward scatter area/side scatter area and forward scatter width/forward scatter area analysis. Live cells (DAPI-) that do not express CD45 and Ter119 were gated and subdivided into PDGFRα-PDGFRβ-(Q3) from where the EC fraction (CD31+, CD13-) is sorted, and PDGFRα-PDGFRβ+ (Q4) from where the pericyte fraction (CD13+, CD31-) is derived. Gating strategies to discriminate cell populations based on their immunolabelling were defined according to reference experiments using single-stained samples and FMO (fluorescence minus one) controls. A figure describing the gating strategy is shown in Supplementary Figure 6c.
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