Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching.

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.

Pericyte dynamics in the mouse germinal matrix angiogenesis.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is the most devastating neurological complication in premature infants. GM-IVH usually begins in the GM, a highly vascularized region of the developing brain where glial and neuronal precursors reside underneath the lateral ventricular ependyma. Previous studies using human fetal tissue have suggested increased angiogenesis and paucity of pericytes as key factors contributing to GM-IVH pathogenesis. Yet, despite its relevance, the mechanisms underlying the GM vasculature's susceptibility to hemorrhage remain poorly understood. To gain better understanding on the vascular dynamics of the GM, we performed a comprehensive analysis of the mouse GM vascular endothelium and pericytes during development. We hypothesize that vascular development of the mouse GM will provide a good model for studies of human GM vascularization and provide insights into the role of pericytes in GM-IVH pathogenesis. Our findings show that the mouse GM presents significantly greater vascular area and vascular branching compared to the developing cortex (CTX). Analysis of pericyte coverage showed abundance in PDGFRß-positive and NG2-positive pericyte coverage in the GM similar to the developing CTX. However, we found a paucity in Desmin-positive pericyte coverage of the GM vasculature. Our results underscore the highly angiogenic nature of the GM and reveal that pericytes in the developing mouse GM exhibit distinct phenotypical and likely functional characteristics compared to other brain regions which might contribute to the high susceptibility of the GM vasculature to hemorrhage.

Deficiency of Notch signaling in pericytes results in arteriovenous malformations.

Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.

PDGFRß-P2A-CreERT2 mice: a genetic tool to target pericytes in angiogenesis.

Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor ß promoter: PDGFRß-P2A-CreER T2 . We evaluated the expression of the PDGFRß-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRß+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRß-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.