In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment
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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 toward 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 multiplicative 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.
KeywordsNotch In vitro Endothelial cell Pericyte Macrophage
The authors would like to thank Carrie Shawber for her consultation and advice on experimental methodology and planning. They are additionally grateful to Warren Pear, Boris Reizis, and Andrew Weng for kind gifts of reagents as detailed in the Materials and Methods. In addition, they would like to thank the Columbia University Medical Scientist Training Program (and its MSTP training grant T32GM007367) for its support.
This study was funded by NIH grants: 1R01HL112626 (J.K.) and 1R01HL119043 (J.K.).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
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