Abstract 224: TRAF-STOP-RHDL-Nanoparticles Reduce Atherosclerosis

Abstract

Background: Inhibition of the costimulatory CD40-CD40L receptor/ligand dyad drastically reduces atherosclerosis. However, its long-term blockage can result in immune suppression. We recently identified small molecule inhibitors that block the interaction between CD40 and TNF Receptor Associated Factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. We investigated the potential of the TRAF-STOPs to treat atherosclerosis. Results: Treatment of ApoE-/- mice with either TRAF-STOP 6877002 or 6860766 reduced both initial and established atherosclerosis and induced a stable plaque phenotype with increased collagen and VSMC content, decreased lipid core, and a decrease in macrophage number. There were no signs of immune suppression or toxicity. In vitro experiments showed that the TRAF-STOPs reduced inflammation in macrophages, but not in T- or B cells, endothelial cells or vascular smooth muscle cells. Intravital microscopy demonstrated that the TRAF-STOPs reduced monocyte recruitment to the plaque. The CD36-mediated uptake of ox-LDL by macrophages and foam cell formation was also inhibited by TRAF-STOPs. Transcriptomics analysis and Ingenuity pathway analysis of TRAF-STOP-treated bone marrow-derived macrophages revealed that the top ranking canonical pathways for both TRAF-STOPs involved pro-inflammatory immune responses and cholesterol biosynthesis. 6877002 also affected cell cycle regulation. Surface plasmon resonance experiments and mutation studies demonstrated that 6877002 and 6860766 had a different interaction site within the TRAF6 C-domain, which explained the additional effect of 6877002. To target TRAF-STOPs specifically to macrophages, 6877002 was incorporated into rHDL nanoparticles. Flowcytometry and fluorescent microscopy demonstrated accumulation of rHDL-6877002 in plaque macrophages after a single dosis. Six weeks of rHDL-6877002 treatment reduced atherosclerosis in ApoE-/- mice. Conclusions: TRAF-STOP 6877002 and 6860766 can overcome the current limitations of long-term CD40 and CD40L inhibition and nanoparticle-mediated delivery TRAF-STOP to plaque macrophages may become a future therapy for atherosclerosis.