Notch Signaling in Vascular Endothelial and Mural Cell Communications

Abstract

The Notch signaling pathway is a highly versatile and evolutionarily conserved mechanism with an important role in cell fate determination. Notch signaling plays a vital role in vascular development, regulating several fundamental processes such as angiogenesis, arterial/venous differentiation, and mural cell investment. Aberrant Notch signaling can result in severe vascular phenotypes as observed in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and Alagille syndrome. It is known that vascular endothelial cells and mural cells interact to regulate vessel formation, cell maturation, and stability of the vascular network. Defective endothelial-mural cell interactions are a common phenotype in diseases characterized by impaired vascular integrity. Further refinement of the role of Notch signaling in the vascular junctions will be critical to attempts to modulate Notch in the context of human vascular disease. In this review, we aim to consolidate and summarize our current understanding of Notch signaling in the vascular endothelial and mural cells during development and in the adult vasculature.

Figure 1.

Schematic of the canonical Notch signaling pathway. (1) Notch receptors (1–4) are initially synthesized as single-chain precursors within the endoplasmic reticulum and subsequently transported to the Golgi apparatus. (2) The Notch precursor then undergoes cleavage by furin forming an extracellular and transmembrane domain and is modified by the glycotransferases fringe. These modified proteins are transported and inserted into the cell membrane. The Notch receptor interacts with Delta/Serrate/Lag (DSL)-2 ligands Jagged 1, Jagged 2, Delta-like 1 (Dll1), Delta-like 3 (Dll3), or Delta-like 4 (Dll4). (3) Upon binding, the Notch receptor is cleaved by ADAM 10 releasing the Notch extracellular domain (NECD) that is then degraded by receptor endocytosis. (4) There is then a subsequent proteolytic cleavage by γ-secretase releasing the Notch intracellular domain (NICD) into the cytoplasm. (5) The NICD is then translocated to the nucleus and forms a coactivator complex with factors including recombination signal-binding protein for immunoglobulin κJ (RBPJK) and Mastermind-inducing transcription of Hes and Hey genes. (Created in BioRender.com.)

Figure 2.

The role of Notch signaling in arterial specification during development. The top panel is a schematic showing how changes in Notch expression impact arterial/venous specification in the arterial/venous network. The two major signaling pathways operate downstream of vascular endothelial growth factor (VEGF)-A, induce arterial differentiation, the Notch pathway, and the PLC/MAPK pathway. In the arterial specification pathway (red arrows), sonic hedgehog–expressing mesodermal cells produce VEGF-A and stimulate endothelial precursor cells. Mesodermal cells expressing sonic hedgehog produce VEGF that interacts with VEGFR2 and neuropilin 1 in arterial precursor cells leading to the expression of Dll4 and Notch. Once an endothelial precursor cell reaches a critical threshold level of Dll4, it causes strong Notch signaling in an adjacent cell and establishes a cell fate decision selecting an arterial over venous endothelial cell fate. There are two distinct mechanisms that drive vein differentiation (blue arrows). The venous endothelial progenitor cells lack neuropilin 1 and low amounts of VEGF trigger expression of chicken ovalbumin upstream promoter transcription factor II (COUP-TFII). COUP-TFII represses VEGFR2, neuropilin 1, and Notch signaling and promotes a venous cell fate and promotes EphB4 expression; additionally, the activation of the PI3K/AKT pathway blocks arterial specification by preventing ERK activation. (Created in BioRender.com.)

Figure 3.

Schematic of Notch signaling in sprouting angiogenesis. (1) Angiogenesis is initiated by a stimulus such as hypoxia, which leads to an increase in vascular endothelial growth factor (VEGF) expression in tissues. (2) The presence of VEGF (green circles) leads to the binding of VEGF receptor 2 (VEGFR2) on the surface of the endothelial cells. A VEGF/Notch-regulated mechanism ensures a limited number of tip cells are formed through a process known as lateral inhibition. After VEGF binds to the VEGFR2 receptor, it promotes the formation of a tip cell (brown cell) and promotes an increase in the Notch ligand Dll4 expression, while simultaneously inhibiting the formation of tip cells by its neighbors through Notch signaling. Activating Notch signaling results in the down-regulation of VEGFR2 and promotes the production of soluble VEGFR (sVEGFR) that can then act to scavenge extracellular VEGF and hence prevent overvascularization. (3) These cells will then become stalk cells that form the body of the sprouting vessel. (Created in BioRender.com.)

Figure 4.

The role of Notch in vascular endothelial and mural cell communication. (A) Schematic of mural cell localization within branching arterioles. (B) Cross-sectional magnification of a capillary highlight pericytes (green), endothelial cells (red), and basal lamina (pink). (C) Transmission electron microscopy (TEM) images of vessels within the retinal ganglion cell layer demonstrate a peg and socket connection between the endothelial cell and pericyte. (D) Notch expressed in the mural cell (green) is activated upon ligand binding from the endothelial cell (red). (E) After a series of proteolytic events (see Fig. 1), the Notch intracellular domain (NICD) translocates to the nucleus and promotes transcription of a number of genes including PDGFRβ, smooth muscle actin (SMA), Notch 3, and Jagged 1, which promote mural cell maturation and stability. (DSL) Delta/Serrate/Lag, (RBPJK) recombination signal-binding protein for immunoglobulin κJ. (Created in BioRender.com.)