Macrophage Expression Of The Notch Ligand Delta-Like 4 Promotes Vein Graft Disease In Ldl Receptor-Deficient Mice

Background: Vein graft failure causes devastating complications in patients with peripheral arterial disease or ischemic heart disease, but its underlying mechanisms remain obscure and no effective therapeutic measures are available. Methods and Results: We tested the hypothesis that Notch signaling triggered by its ligand Delta-like 4 (Dll4) promotes macrophage activation and vein graft disease. 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. [Approach 1: Dll4 antibody] Dll4 blocking antibody was administered for 28 days. Dll4 blockade inhibited lesion development and macrophage accumulation (Figures) in vein grafts, and suppressed macrophage expression of genes typical of pro-inflammatory M1 macrophages (e.g., IL-1β, TNF-α). In vivo molecular imaging demonstrated that Dll4 antibody treatment suppressed MMP activity in lesional macrophages. Dll4 blockade concomitantly attenuated collagen thinning. [Approach 2: siRNA delivery to macrophages] To address the relative contribution of macrophages to Dll4-mediated vein graft disease in vivo, we delivered Dll4 siRNA oligos encapsulated in macrophage-targeted lipid nanoparticles. In vivo Dll4 silencing in macrophages reduced lesion development and macrophage burden in vein grafts of Ldlr-/- mice to a similar extent as those of Dll4 antibody therapy. In vitro gain-of-function and loss-of-function studies suggested that Dll4 promotes expression of pro-inflammatory molecules in macrophages. Furthermore, macrophage Dll4 stimulated smooth muscle cell (SMC) proliferation and migration and suppressed their differentiation. Conclusions: These results suggest the novel mechanism that macrophage Dll4 promotes vein graft lesion development by exacerbating inflammation and crosstalk between macrophages and SMC, supporting the Dll4-Notch axis as a potential therapeutic target.