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Abstract

Pericytes are mural cells of the microvascular system of mesenchymal origin, which abluminally ensheath capillary endothelial cells (EC). Pericytes play a pivotal role in the maturation of newly formed vessels and are a hallmark of the quiescent endothelium in most microvascular beds. Pericyte-based research is hampered by the limited availability of robust pan-pericyte markers due to their overlapping marker expression with various cells of mesenchymal origin. In order to identify novel functionally relevant pericyte markers, an expression profiling of five different human primary pericyte populations and other mesenchymal cell populations (EC, adipocytes, fibroblasts, mesenchymal stem cells [MSC]) was performed. Among others, this screen identified Prostaglandin E receptor 2 (PTGER2) and Sphingosine-1-phosphate receptor 3 (S1PR3), two Gprotein-coupled receptors (GPCRs), as novel, highly pericyte-enriched transcripts. Only S1PR3 showed comparable expression levels also in smooth muscle cells (SMC). Subsequent cellular studies demonstrated for the first time that S1PR3 signals via Gαi and Gαq in pericytes and regulates myosin light chain (MLC) phosphorylation. To study the role of pericyteexpressed S1PR3 in a physiological setting, comparative co-culture experiments of EC with S1PR3silenced pericytes were performed and transcriptomic profiles were traced. The expression of S1PR3 by pericytes resulted in subtle, but distinct transcriptomic changes, including changes in cell-cell as well as cell-extracellular matrix interaction molecules. MPRIP, a regulator of RhoA and MLC phosphorylation, was identified as one of the most promising candidate genes. Functional in vitro assays of S1PR3 silenced pericytes resulted in reduced transmigration capacity and increased cell size. Notably, PTGER2 was exclusively expressed by pericytes. Transcriptomic analyses of co-cultured pericytes silenced for PTGER2 revealed expression changes of proliferation-associated genes (downregulation of negative regulators). Correspondingly, functional in vitro assays of PTGER2 silenced pericytes resulted in enhanced proliferation. To enable the validation of pericyte-expressed S1pr3 and Ptger2 and further functional studies in vivo, mouse models for the conditional deletion of these genes are required. Based on the expression profiling and cellular screening experiments and the fact that conditional mice for S1pr3 are not available, experiments were set out with the aim to generate S1pr3 floxed mice by CRISPR/Cas technology. Sequential integration of two LoxP sites using Cas9 wildtype was successfully used to generate conditional S1pr3 mice. Taken together, the experiments identified PTGER2 as novel pericyte marker and S1PR3 as novel mural cell marker within the mesenchymal cell lineage that both control important pericyte functions. Furthermore, CRISPR/Cas technology proved as a suitable approach to generate conditional S1pr3 knockout mice.