Molecular characterization of vascular malformations using single-cell RNA sequencing and immunohistological analysis

Abstract: CCM is a human disease in which vascular malformations develop in the central nervous system, predominantly in the brain. The malformations which can measure up to several centimeters in size are dilated and leaky vessels, with unstable cellular junctions and thin vessel walls. Depending on the size and the location of the malformation, the lesion might impose a direct threat to the health of the patient. The symptoms vary from more mild health issues such as headaches, to more severe symptoms such as loss of vision or hearing, or even epileptic seizures, paralysis, and hemorrhagic strokes. CCM is caused by a mutation in either of the CCM genes: CCM1, CCM2, or CCM3. The molecular mechanisms underlying the development of the vascular malformations are still poorly understood, but it has recently been shown that the GTPase CDC42 interacts with the CCM protein complex. By inducing endothelial cell-specific deletion of Cdc42, similar malformations to that of CCM arise in the mouse brain. In this project, our aim was to investigate the molecular changes underlying the vascular malformations caused by endothelial specific Cdc42 deletion in mice. To do so, we isolated endothelial cells from Cdc42iΔEC and Control mouse brain at postnatal day seven and performed single-cell RNA sequencing of these cells. When analyzing the data, we noticed that a subset of KO cells clustered differently and showcased a unique molecular profile. These cells expressed pan-endothelial markers but did not classify as arteries, capillaries, or veins as they did not express markers for those endothelial sub-types. Instead, these cells expressed markers for fenestrated choroid plexus endothelium, such as Igfbp3, and Gpihbp1, yet the absence of Aqp1 expression distinguishes them from choroid plexus endothelial cells. To validate the results from the RNA sequencing experiments we performed immunohistochemistry on brain sections of Cdc42iΔEC and Control mice.   In summary, we identified a unique expression pattern in a subset of Cdc42 depleted endothelial cells. While further investigations are needed, this is an important starting point for the identification of potential biomarkers, drug targets and the development of urgently needed treatment strategies.