Background Vein graft failure is a global health burden with no effective medical solutions. Saphenous vein grafts for peripheral artery disease are widely used because they remain patent longer than artificial conduits. Approximately 50% of vein grafts for lower extremity arterial disease, however, become occluded or narrowed within a year, leading to devastating limb amputation or expensive angioplasty or surgical revascularization. Although many mechanisms for arterial diseases have been established, the pathogenesis of vein graft failure remains incompletely understood. We previously demonstrated that Notch signaling triggered by its ligand Delta-like 4 (Dll4) induces macrophage activation (1,2). We tested the hypothesis that macrophage Dll4 promotes the development of vein graft disease. Methods Vein graft surgery was performed in high-fat fed LDL receptor-deficient (Ldlr-/-) mice by implanting donor inferior vena cava into recipient right carotid arteries under general anesthesia (ketamine 90 mg/kg, xylazine 10 mg/kg, i.p.). We used clinically relevant biotherapeutics: 1) Dll4 blocking antibody; and 2) macrophage-targeted Dll4 siRNA. Statistical analysis of differences was performed by Student’s t test. Results 1) Antibody Dll4 antibody or control IgG (250 mg; n=9-10) was administered for 28 days. Dll4 antibody treatment inhibited lesion development (Figure 1) and macrophage accumulation (Mac3 immunostain, Figure 2) in vein grafts. Dll4 blockade suppressed macrophage expression of genes typical of pro-inflammatory “M1” macrophages (e.g., IL-1β, TNFα). In vivo molecular imaging of lesional macrophages demonstrated that Dll4 antibody suppressed the activity of MMPs, matrix-degrading enzymes responsible for plaque vulnerability. Dll4 blockade attenuated collagen thinning, suggesting the lesion stabilization. 2) siRNA To address the relative contribution of macrophages to Dll4-mediated vein graft disease in vivo, we delivered Dll4 siRNA or control siRNA (0.5 mg/kg; n=8-9) encapsulated in macrophage-targeted lipid nanoparticles. In vivo Dll4 silencing in macrophages reduced lesion size (p<0.05) and macrophage burden (p<0.05) in vein grafts of Ldlr-/- mice to a similar extent as those of Dll4 antibody therapy. 3) In vitro studies In gain-of-function and loss-of-function experiments, Dll4 promoted pro-inflammatory molecules (e.g., “M1” genes) in macrophages. Macrophage Dll4 stimulated smooth muscle cell proliferation and migration and suppressed their differentiation. Conclusion These results suggest the novel mechanism that macrophage Dll4 promotes vein graft lesion development by exacerbating inflammation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4-Notch axis as a potential therapeutic target.