In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment

Abstract

Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals, and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed “angiotrophism”. We found that polarizing macrophages towards a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes, and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has additive effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.

Altered feto-placental vascularization, feto-placental malperfusion, and fetal growth restriction in mice with Egfl7 loss-of-function

“…To determine whether Egfl7 plays a role in placental EC sprouting, we performed a fibrin gel bead assay, an in vitro model that recapitulates key early stages of new vessel formation in a threedimensional matrix, including vessel sprouting and maturation (Nehls and Drenckhahn, 1995;Tattersall et al, 2016;Nakatsu and Hughes, 2008). Egfl7 −/− placental ECs formed significantly fewer and shorter endothelial sprouts when compared with control placental ECs at day 2 and day 4 in culture ( Fig.…”

Asymmetry at cell-cell interfaces direct cell sorting, boundary formation, and tissue morphogenesis

“…Interestingly, inhibition of Notch signaling can downregulate the expression of proangiogenic genes ephrin-B1 and Sox17 at the transcriptional and post-transcriptional levels, respectively [81] [82] (Figure). Also, Notch activity has been shown to be crucial for transmitting differentiation, proliferation, survival, and angiogenesis signals to endothelial cells [83] [81] [84]. Thus, it is not surprising that homozygous mutations in Notch show embryonic lethality in mice in combination with vascular remodeling defects [85] [86].…”

Notch, lipids, and endothelial cells

“…For example, in the liver, Dlls expressed by synovial endothelial cells activate Notch signaling in Th1 lymphocytes, inducing the expression of IL-10 to blunt the inflammatory response [70 ■ ]. Interactions between macrophage Notch and Dll4 expressed on tip-cells are important for retinal sprouting angiogenesis [71] and recently an in vitro model of angiogenesis integrated Notch/ligand interactions in macrophages, mural cells, and endothelial cells [72 ■ ]. Finally, Notch activation in smooth muscle cells, essential for their fate decision and maintenance, is driven in part by ligands expressed by endothelial cells [73] (Fig.…”