Temporal and embryonic lineage-dependent regulation of human vascular SMC development by NOTCH3

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8-9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.