Abstract 105: Smooth Muscle Cell-derived Vascular Progenitor Cells Promote Arterial Remodeling and Fibrosis Through Loss of Hedgehog/Wnt/β-catenin/Klf4 Activity

Resident vascular adventitial progenitor cells express the stem cell marker, Sca1 (AdvSca1 cells). Using smooth muscle cell (SMC) lineage tracing models and YFP reporter knock-in, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) that originate from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. The adventitial microenvironment and induction of the transcription factor, Klf4 is critical to reprogramming; however, the mechanism of Klf4 induction is unknown. We aimed to define the signaling pathways involved in SMC reprogramming and to define the fate of AdvSca1-SM cells in response to vascular injury. Flow sorting was used to isolate YFP + Sca1 - SMCs, YFP + Sca1 + AdvSca1-SM cells, and YFP - Sca1 + non-SMC-derived AdvSca1 cells from aortae (AO) and carotid arteries (CA) of SMC YFP reporter mice. RNA was subjected to RNA-seq analysis. Unbiased hierarchical clustering revealed that genes related to hedgehog/Wnt/β-catenin signaling are significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in SMC reprogramming to AdvSca1-SM cells. CA injury was induced by ligating the left common CA of SMC YFP reporter mice. Using RNA-seq, we found that in response to injury, AdvSca1-SM cells downregulate genes in the hedgehog/Wnt/β-catenin signaling pathway, as well as stemness-related genes, and adopt a myofibroblast-like phenotype. To selectively fate-map AdvSca1-SM cells, we took advantage of the high and selective expression of Gli1 by AdvSca1-SM cells and generated Gli1-CreER T2 -Rosa26-YFP reporter mice. Flow Cytometry and Immunofluorescent (IF) staining show that YFP was expressed exclusively in adventitial Sca1-positive cells, but not all adventitial Sca1-positive cells expressed YFP. RNA-seq analysis confirmed selective upregulation of hedgehog/Wnt signaling in YFP + Sca1 + cell population compared to the YFP - Sca1 + population, supporting this model to faithfully track AdvSca1-SM cells. CA injury induced adventitial expansion and differentiation of AdvSca1-SM cells, as indicated by increased YFP + cells and decreased Sca1 expression. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis, but not neointima formation.