We characterize a sonic hedgehog (Shh) signaling domain restricted to the adventitial layer of artery wall that supports resident Sca1-positive vascular progenitor cells (AdvSca1). Using patched-1 (Ptc1 lacZ ) and patched-2 (Ptc2 lacZ ) reporter mice, adventitial Shh signaling activity was first detected at embryonic day (E) 15.5, reached the highest levels between postnatal day 1 (P1) and P10, was diminished in adult vessels, and colocalized with a circumferential ring of Shh protein deposited between the media and adventitia. In Shh ؊/؊ mice, AdvSca1 cells normally found at the aortic root were either absent or greatly diminished in number. Using a Wnt1-cre lineage marker that identifies cells of neural crest origin, we found that neither the adventitia nor AdvSca1 cells were labeled in arteries composed of neural crest-derived smooth muscle cells (SMCs). Although AdvSca1 cells do not express SMC marker proteins in vivo, they do express transcription factors thought to be required for SMC differentiation, including serum response factor (SRF) and myocardin family members, and readily differentiate to SMC-like cells in vitro. However, AdvSca1 cells also express potent repressors of SRF-dependent transcription, including Klf4, Msx1, and FoxO4, which may be critical for maintenance of the SMC progenitor phenotype of AdvSca1 cells in vivo. We conclude that a restricted domain of Shh signaling is localized to the arterial adventitia and may play important roles in maintenance of resident vascular SMC progenitor cells in the artery wall.artery ͉ differentiation ͉ Klf4 ͉ self-renewal ͉ serum response factor

The arterial adventitia plays important roles in growth and remodeling of the artery wall. We previously reported that sonic hedgehog (Shh) signaling is restricted to the adventitial layer of large and medium‐sized arteries in the perinatal period. Localized within this adventitial domain we found a resident population of Sca1‐positive cells (AdvSca1). Although AdvSca1 cells do not express smooth muscle cell (SMC) markers in vivo, they do express SRF and myocardin, and differentiate into SMCs when removed from the adventitia and placed in cell culture. AdvSca1 cells also express potent SRF‐dependent corepressors including KLF4 and Msx1, which may play critical roles in maintaining the SMC progenitor phenotype. We have tested this possible role of KLF4 by gain and loss of function approaches in vitro. Knockdown of KLF4 expression by siRNA results in a loss of Sca1 expression, consistent with a loss of self‐renewal potential of AdvSca1 cells. Conversely, exposure of AdvSca1 cells to KLF4 adenovirus inhibited SMC differentiation. By seven days after >80% flow reduction in a mouse carotid model, a substantially thickened neoadventitia formed that contained increased numbers of AdvSca1 cells with no evidence of SMC marker upregulation. These results suggest that resident AdvSca1 cells can proliferate in response to a stimulus for arterial remodeling, and that KLF4 plays important roles in AdvSca1 cell self‐renewal.

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