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. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has additive effects on sprouting. 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. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. 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. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed “angiotrophism”. 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. Multicellular interactions during angiogenesis are difficult to study in animals, and challenging in a reductive setting. Experiments were performed once.Īngiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. d Quantification of sprout number in both experiments. c Dll4KD HUVEC show increased sprouting in endothelial and pericyte coculture capillary sprouting assay. b Dll4KD HUVEC show increased sprouting in endothelial cell-only capillary sprouting assay. a Protein expression of Dll4 in cells co-infected with RFP and the Dll4KD shRNA construct are greatly reduced compared to RFP/Scramble control. Dll4 inhibition increases sprouting in endothelial cell-only capillary sprouting assays and in endothelial-pericyte cocultures. Online Resource 8 Knockdown of Jagged1 in pericytes reduces cell growth in monoculture. Protein expression of Jagged1 in cells co-infected with RFP and the Jagged1KD shRNA construct are approximately half that of RFP/Scramble controls. Online Resource 7 Pericyte/endothelial knockdown of Jagged1 qPCR was validated via western blot. HBVP resuspension in fibrin gel, rather than direct binding to the endothelial-coated beads, does not produce the profound phenotypic changes observed in the case of direct pericyte association with endothelial cells. Pericyte dispersal in fibrin gel does not recapitulate effects of direct bead binding. LysM cre/+ control mice do not express GFP. Bone marrow macrophages derived from LysM cre/+ DNMAML-GFP fl/+ mice show widespread expression of GFP, indicating synthesis of the DNMAML-GFP gene product. Macrophage expression of DNMAM-GFP was confirmed via FACS. No relationships are statistically significant for p<0.05. a Treatment with 0.1, 1.0, or 10 ng/mL LPS did not significantly alter sprout number or tip cell number. LPS treatment does not alter angiogenesis in endothelial cell-only capillary sprouting assays. Myeloid cells treated with proinflammatory factors LPS and IFNγ show increased mRNA expression of pro-inflammatory marker iNOS/NOS2, while cells treated with alternative activator cytokine IL4 show increased mRNA expression of alternative activation marker Arginase. Macrophage polarization was confirmed via qPCR. d Quantification of frequency of longer (>200um) sprouts. b Sprout number and length remains increased through late timepoint day 5. a EOC2 inclusion increases number and length of endothelial sprouts at early timepoint day 3. b Schematics of capillary sprouting assays incorporating bone marrow macrophages (directly into the fibrin matrix) and pericytes (bound to the bead on top of the endothelial monolayer). a Schematic of an endothelial cell-only capillary sprouting assay, involving coculture of HUVEC with an overlying D551 fibroblast feeder layer. Schematic of the incorporation of macrophages and pericytes in the capillary sprouting assay.
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