Gene Regulation by RNP Granule Formation in Atherosclerosis

New York University School Of Medicine, New York NY

Investigators

Abstract

PROJECT SUMMARY Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages in the arterial intima. A better understanding of the role ribonucleoprotein (RNP) granules play in regulating the innate immune system, may reveal important mechanisms underlying this maladaptive inflammatory response in atherosclerosis. We hypothesize that macrophage transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SGs). SG formation is increased in plaque macrophages of atherosclerotic mice and can be induced in vitro during cellular cholesterol loading. To test our hypothesis, we using immunohistochemistry observed that the SG protein, G3BP1, is significantly increased in progressing plaques compared to regressing. Using centrifugation, we isolated SGs from bone marrow derived macrophages treated with atherogenic stimuli, and found enrichment of cholesterol efflux transcripts within the SG, suggesting that SGs play a role in cholesterol homeostasis. Using imaging and RNA-seq of macrophages treated with siG3bp1 and Dil-oxLDL, we found that G3bp1 knockdown reduced lipid binding, uptake, macrophage phagocytic capacity, and efflux. Altogether, these data support a role for SGs in dysregulating cholesterol pathways in plaque macrophages during atherosclerosis progression. In this proposal, I aim to characterize stress granule formation in macrophages exposed to atherogenic stimuli in vitro and identify the RNA transcripts and RNA-binding proteins sequestered. By investigating post-transcriptional modifications, I will uncover the mechanism by which transcripts are marked for sequestration or escape from SGs. Additionally, I will use in vivo mouse models of atherosclerosis to determine the temporal kinetics, cellular localization, and quantity of SGs per cell over disease course. I aim to test the therapeutic potential of targeting SGs by perturbing SG formation using (1) G3BP1fl/fl iLysMcre (genetic) and (2) Ldlr–/– with a small molecule inhibitor of SG formation, ISRIB (therapeutic), in atherosclerotic mice and characterize plaque burden. Collectively, the proposed investigation into RNP granules in atherosclerosis, will reveal novel mechanisms of post-transcriptional and innate immune regulation, and provide foundational studies for new therapeutic strategies.