Objective Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. Methods and Results Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro towards natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Dll4 ligand or constitutively active Notch receptors, and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted VE-cadherin and β-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrate that Notch activation increases myosin light chain (MLC) phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced MLC phosphorylation, and led to enhanced barrier function by reorganizing F-actin to β-catenin containing cell-cell adherens junctions. Conclusions Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells trigger a senescence phenotype associated with endothelial barrier dysfunction.

Rationale Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. Objective Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared to uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMC. Methods and Results In human primary VSMC, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27kip1 were observed downstream of Jag-1/Notch2 signaling, and required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination and prolonged half-life of p27kip1. Jag-1/Notch2 signaling robustly decreased S-phase kinase associated protein (Skp2), an F-box protein that degrades p27kip1 during G1. Over expression of Skp2 prior to Notch activation by Jag-1 suppressed the induction of p27kip1. Additionally, increased Notch2 and p27kip1 expression was co-localized to the non-proliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen (PCNA), whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation. Conclusions We propose a receptor specific function for Notch2 in regulating Jag-1-induced p27kip1 expression and growth arrest in VSMC. During vascular remodeling, co-localization of Notch2 and p27kip1 to the non-proliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus Notch2 is a potential target for control of VSMC hyperplasia.

Objective BMP9/ALK1 and DLL4/Notch promote endothelial quiescence, and we aim to understand mechanistic interactions between the two pathways. We identify new targets that contribute to endothelial quiescence, and test whether loss of Dll4+/− in adult vasculature alters BMP signaling. Approach and Results Human endothelial cells respond synergistically to BMP9 and DLL4 stimulation, showing complete quiescence and induction of HEY1 and HEY2. Canonical BMP9 signaling via ALK1-Smad1/5/9 was disrupted by inhibition of Notch signaling, even in the absence of exogenous DLL4. Similarly, DLL4 activity was suppressed when the basal ALK1-Smad1/5/9 pathway was inhibited, showing these pathways are interdependent. BMP9/DLL4 required induction of P27KIP1 for quiescence, although multiple factors are involved. To understand these mechanisms, we used proteomics data to identify upregulation of thrombospondin-1, which contributes to the quiescence phenotype. To test whether Dll4 regulates BMP/Smad pathways and endothelial cell phenotype in vivo, we characterized the vasculature of Dll4+/− mice, analyzing endothelial cells in the lung, heart, and aorta. Together with changes in endothelial structure and vascular morphogenesis, we found that loss of Dll4 was associated with a significant upregulation of pSmad1/5/9 signaling in lung endothelial cells. Since steady state endothelial cell proliferation rates were not different in the Dll4+/− mice, we propose that the upregulation of pSmad1/5/9 signaling compensates to maintain endothelial cell quiescence in these mice. Conclusions DLL4/Notch and BMP9/ALK1 signaling rely on each other’s pathways for full activity. This represents an important mechanism of crosstalk that enhances endothelial quiescence and sensitively coordinates cellular responsiveness to soluble and cell-tethered ligands.