Abstract 220: Targeting Cholesterol Efflux, Autophagy, and Inflammation to Prevent Atherosclerosis

Introduction: The NLRP3 inflammasome and Toll-like receptor signaling is activated in advanced human atherosclerotic plaques, while autophagy and reverse cholesterol transport (RCT) become dysfunctional. Simultaneous targeting of these pathways can prevent CVD. Objectives: To determine if Miltefosine can prevent atherosclerosis by inducing RCT/autophagy and dampening inflammation. Methods and results: Here, we report that Miltefosine, an anti-leishmanial drug, acts to decrease atherosclerosis in vivo, and that it induced RCT/autophagy while dampening TLR signaling and NLRP3 inflammasome activity. Miltefosine treatment of macrophages disrupted lipid rafts; ~26 % decrease vs. control via alexa647-CTB binding by flow-cytometry, (p<0.005, n=4), and increased ABCA1 mediated cholesterol efflux to apoA1; ~20% increase in treated vs. control (p<0.001, n=3). Macrophages treated with Miltefosine vs. control exhibited a marked increase in autophagosomes, indicated by p62 and LC3 staining puncta. Autophagic degradative flux was not inhibited by Miltefosine. Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester:free cholesterol) ratio (p<0.005, n=3). Miltefosine treated vs. control macrophages showed ~75% reduction in pro-IL1β mRNA levels upon LPS induction (p<0.05, n=3). Miltefosine potently inhibited NLRP3 inflammasome assembly upon LPS/ATP treatment leading to ~70% reduction in ASC1 speck positive cells. Gasdermin D mediated release of mature IL1β was reduced by ~80% in Miltefosine treated vs. controls (p<0.01, n=3), while only ~20% reduction was observed by cyclodextrin treatment, indicating that inflammasome inhibition by Miltefosine was not only due to cholesterol depletion. In vivo pilot studies showed that Miltefosine added to a chow diet increased RCT to plasma (~70% increase in 24h vs. control), and feces (~60% increase in 24h vs. control, p< 0.005, n=5) in C57BL6 mice. Atherosclerotic lesions were =50% smaller in apoE -/- mice fed chow diet + Miltefosine vs. controls (p<0.02, n=5). Conclusion: Miltefosine induced RCT and autophagy while dampening TLR signaling and NLRP3 inflammasome activity, and it decreased atherosclerosis lesion burden in mice.