Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

CXCR3-CXCL11 signaling restricts angiogenesis and promotes pericyte recruitment.

Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces, yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs-the chemokine receptor, CXCR3, and one of its ligands, CXCL11-that delimits EC angiogenic potential and suppresses pericyte recruitment during development through regulation of pdgfb expression in ECs. In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with and expansion of the vasculature in zebrafish treated with the Cxcr3 inhibitor AMG487. We also demonstrate using flow modeling platforms that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared to their control counterparts. Together these data suggest that CXCR3 signaling in ECs drives vascular stabilization events during development.