PROJECT ABSTRACT A maladaptive inflammatory response to lipid imbalance underlies the chronic vascular inflammation in atherosclerosis. Persistent activation of the Integrated Stress Response (ISR) signaling is also observed in both mouse and human atheroma. ISR is an elaborate, homeostatic signaling activated by a range of conditions such as hypoxia, hyperlipidemia and endoplasmic reticulum (ER) and mitochondrial stress, which are known to promote atherosclerosis. Small molecules and genetic models that prevent hypercholesterolemia-induced ISR signaling were shown to prevent atherosclerosis progression, demonstrating ISR’s causality in atherosclerosis development. Despite regulating lipid-induced sterile inflammation, thereby representing a novel therapeutic opportunity in cardiovascular disease (CVD), therapeutic targeting of a homeostatic pathway such as ISR in a chronic disease is not without its challenges. Deciphering the detailed mechanisms by which ISR governs macrophage immunometabolism and atherogenesis can pave the way to effective and specific therapeutic strategies in CVD while escaping toxicity that may be associated with targeting homeostatic signaling. We made the striking discovery that ISR inhibition in hypercholesterolemic mice leads to increased 5- hydroxymethylcytosine (5-hmC) to 5-methylcytosine (5-mC) ratio in macrophages and plaques while reducing IL-1b and atherosclerosis progression. The oxidation of 5-mC to 5-hmC is catalyzed by Ten eleven translocation (TET) family of methylcytosine dioxygenases, somatic mutations in which are associated with coronary heart disease and early-onset myocardial infarction (MI). The inactivation of TET-2 in mice promotes atherosclerosis progression and cardiac dysfunction. Our robust preliminary data shows that hypercholesterolemia induces 2- hydroxyglutarate (2HG), a potent TET inhibitory metabolite, in an ISR-dependent manner. Furthermore, supplementation with a-ketoglutarate (aKG), a cofactor for TET, stimulates TET activity while inhibiting IL-1b secretion in mouse and human macrophages. aKG supplementation in a small group of hypercholesterolemic mice prevented inflammation while reversing TET inhibition. Building on the insight gained through our robust preliminary studies and incorporating additional evidence from literature, we hypothesize that hyperlipidemia- induced ISR signaling generates an immunometabolite that can promote macrophage inflammatory response and atherosclerosis. We propose to investigate ATF4’s role in regulating macrophage immunometabolism and promoting atherosclerosis in vivo. We will also investigate the consequences of modulating 2HG levels in myeloid cells on inflammation and atherosclerosis in hypercholesterolemic mice. The completion of the proposed studies will illuminate the metabolic and epigenetic consequences for ISR signaling in macrophages on sterile inflammation and atherosclerosis development. The new knowledge gained through these studies could p...