Abstract Body: Introduction. Piezo1 is a mechanosensitive ion channel that gates calcium and sodium influx in response to membrane deformation or elevated shear stress. We previously found that the chronic loss of endothelial Piezo1 leads to reduced cerebral blood flow, brain inflammation, and alterations in neurobehavioral tests. The present study was designed to better define the functional role of endothelial Piezo1 by determining the changes in the brain transcriptional landscape in response to selective Piezo1 loss-of-function in endothelial cells (EC).
Methods. EC-specific inducible Piezo1 loss-of-function (LOF) mice (Cdh5(Pac)CreERT2;Piezo1 fl/fl) or control mice (Piezo1 fl/fl only) were injected with tamoxifen (75 mg/kg). After one month, brains were harvested for single-cell RNA sequencing (scRNA-seq) (10X Genomics) and analysis (SeqGeq V1.7, BD Biosciences).
Results. We observed a broad differential distribution pattern across 10 principal brain clusters between control and EC Piezo1 LOF groups. Gene ontology (GO) analysis showed neuron, microglia, and astrocyte clusters associating with locomotory behavior, learning and memory, and gliogenesis. The EC cluster was linked to gliogenesis, while pericyte and mixed oligodendrocytes/ependymal cell clusters linked with GO terms associated with learning and memory. To explore whether EC Piezo1 LOF was linked to the emergence of specific endothelial cell population, we further sub-clustered the EC population. We found an endothelial cell subtype defined by Igf1r+ and Ptprg+ that was significantly increased (+36.9 %) in the EC Piezo1 LOF brain. Meta-analysis of DEGs in this population revealed their involvement in cell junction organization, fluid shear stress, atherosclerosis, and reactive oxygen species metabolism, while GO analysis highlighted "multicellular organismal response to stress" encompassing genes related to locomotory behavior, learning, memory, and cognition, and fear response behavior. Lastly, this EC population showed an association with the reactive oxygen species metabolic process and the Hif1 signaling pathway.
Conclusions. In addition to promoting reduced cerebral perfusion, the specific loss of endothelial Piezo1 function causes widespread transcriptome alterations within the brain. Within the EC population, Piezo1 LOF results in a notable stress response with links to organismal dysfunction. These studies emphasize the functional importance of EC Piezo1 signaling in brain health.