Bar graphs show results of real-time PCR. no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces pro-inflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell (SMC) proliferation and migration and suppressed their differentiation. Conclusion These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and SMC, supporting the Dll4-Notch axis as a novel therapeutic target. strong class=”kwd-title” Keywords: bypass grafting, inflammation, antibody, RNAi, biotherapy Introduction Vein graft failure is a global health burden with no effective medical solutions.1 AGN 210676 Due to the pandemic of atherosclerotic peripheral artery disease (PAD) and the growing prevalence of underlying metabolic disorders,2 the incidence of vein graft failure is rising. Although many mechanisms for arterial diseases have been established, the pathogenesis of vein graft failure remains incompletely understood. Autologous saphenous vein grafts (SVG) are widely used for PAD because they remain patent longer than artificial conduits.3 AGN 210676 Approximately 50% of lower extremity SVG, however, become occluded or narrowed within a year.4 When PAD grafts fail, the only available therapeutic options are devastating limb amputation or invasive and expensive angioplasty or surgical revascularization. Coronary artery SVG also fail at high rates.5 Although current therapies such as statins can reduce the onset of complications of arterial diseases (e.g., myocardial infarction),6 no effective medical solutions are available for vein graft failure. The AGN 210676 Notch pathway, involving ligands (Delta-like ligand 1 [Dll1], Dll3, Dll4, Jagged1, Jagged2) and receptors (Notch1-4), contributes to biological processes during development and to disease mechanisms in adults.7, 8 Direct cell-to-cell contract via the binding of a ligand to a Notch receptor, both of which are expressed on the cell surface, triggers downstream responses.9 We previously demonstrated that Dll4-mediated Notch signaling promotes macrophage activation.10, 11 Clinical and preclinical evidence has established the causal role of macrophages in arterial atherosclerosis.12, 13 Failing vein grafts also tend to contain macrophages,2, 14 but their role in the disease progression remains unclear. To test the hypothesis that macrophage Notch signaling contributes to the pathogenesis of vein graft disease, the present study used two clinically-relevant biotherapeutics: 1) Dll4 blocking antibody; and 2) Dll4 siRNA encapsulated in macrophage- or endothelial cell (EC)-targeted lipid nanoparticles (LNP). Materials and Methods Materials and Methods are available in the online-only Data Supplement. Results Increased expression of Dll4 in macrophages in human and mouse vein grafts In control human saphenous veins before grafting, little if any intimal cells were immunoreactive for Dll4, whereas the thickened intima of failed human SVG contained many cells expressing Dll4 (Figures 1A; Supplemental Figure I). In the failed grafts, some CD68-positive intimal macrophages were immunoreactive to Dll4 antibody (Supplemental Figure IB). In high-cholesterol/high-fat-fed Ldlr?/? mice, IVC implanted into the carotid artery developed more advanced AGN 210676 lesions than in wild-type mice.15 The neointima of vein grafts in Ldlr?/? mice showed features similar to those of advanced arterial plaques prone to rupture, including foam cell accumulation, microvessels, and intraplaque hemorrhage (Supplemental Figure II), supporting previous reports on a similar model in hypercholesterolemic ApoE3*Leiden mice by the Paul Quax group.16, 17 Vein grafts of Ldlr?/? mice expressed higher levels of Dll4 mRNA compared to native IVC of Ldlr?/? or wild-type mice (qPCR, Figure 1B). In mouse vein grafts, Dll4 localized primarily to intimal macrophages, while smooth muscle cell (SMC) expression of Dll4 was minimal (Day 28, double immunofluorescence, Figure 1C). Ligand binding promotes the cleavage of Notch receptors and release of the intracellular domain.9 The amount of Notch1 intracellular domain (NICD), as identified by the antibody that recognizes the neoepitope, thus indicates TNF-alpha the levels of Notch signaling activation. NICD accumulated primarily in intimal macrophages of vein grafts 28 days after implantation, while few if any smooth SMC and EC were stained positively (Supplemental Figure III). Dll4 and NICD were almost undetectable in the native IVC (Supplemental Figure IVA). But the amounts of immunoreactive Dll4 and NICD in the intima of mouse vein.