Abstract 3942: Identification Of Endothelin Type-A Receptor-expressing Cells Involved In Embryonic Brain Angiogenesis

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Interaction between endothelial cells (ECs) and mural cells (pericytes/smooth muscle cells) is crucial for vascular stabilization and maturation during the late phase of angiogenesis. However, underlying mechanisms and its role in the early sprouting angiogenesis remain largely unveiled. Recently, we have established mice in which EGFP is knocked-in into the endothelin type-A receptor (EdnrA) locus by Cre recombinase-mediated cassette exchange method (RMCE), resulting in the visualization of EdnrA-expressing cells including neural crest (NC)-derived ectomesenchymal cells. We found sporadic distribution of EGFP+cells also in the neural tube (NT) after E9.5, when sprouting angiogenesis into the brain starts. We then examined possible involvement of these cells in angiogenesis. EGFP+cells were first observed between E9.5 to E10.0 in the NT close to its outer surface. Thereafter, the cells tended to distribute toward the ventricular zone. Interestingly, most EGFP+cells in the NT were associated with forming vessels marked by CD31. At E9.5, when migrating CD31+cells were penetrating into the NT, many sprouts were accompanied by EGFP+cells. EGFP+cells expressed PDGFRβ, a marker for mesenchyme-derived mural cells, whereas the cells were negative for CD31, PDGFRα, GFAP, nestin and Iso-B4-lectin binding. At the late gestation stage (E18.5), EGFP+cells formed a layer underlying ECs and became partly positive for α-smooth muscle actin. These results are consistent with the previous finding that mural cells of the forebrain blood vessels originate from the NC, and indicate that CD31+EC/angioblasts and EGFP+immature cells may coordinately give rise to vascular networks in the brain from the early angiogenic stage. It is also noteworthy that EGFP+cells were closely associated with angiogenic ECs in aortic ring assay and in vivo ischemic lesion in the mouse hindlimb. Although these EGFP+cells are not apparently of NC origin, the identification of EdnrA-expressing cells may clarify mechanisms for mural cell differentiation and cell-to-cell interaction involved in angiogenesis. Furthermore, the RMCE knock-in system enables us to unveil the secrets of mural cells in angiogenesis by various genetic modifications.