SUMMARY: Vascular smooth muscle cells (VSMCs) in the blood vessel wall play pivotal roles in cardiovascular disease (CVD; e.g., hypertension), which affects ~48% of US adults, and which is significantly accelerated by diabetes (DCVD). When stimulated by factors like diabetes and the growth factor Angiotensin II (AngII), mature VSMCs de-differentiate through “phenotypic switching” (Phe-sw) via dysregulation of "contractile" and "synthetic” genes, resulting in increased VSMC proliferation, migration, inflammation, and extracellular matrix (ECM) production. In the previous funding cycles, we unraveled the first functional roles for AngII-regulated long noncoding RNAs and (super-)enhancers (a key epigenetic regulatory layer) in VSMCs. Our overall objective in this renewal is to elucidate the mechanisms regulating diabetes- and AngII-induced VSMC Phe-sw at the epigenome and single-cell (sc) level and how these mechanisms establish vascular metabolic memory (in which prior hyperglycemia/AngII exposure leads to persistent long-term DCVD despite subsequent glucose normalization). We will use state-of-the-art multi-omics and sc-sequencing (seq) approaches to decipher VSMC behavior in DCVD and metabolic memory and identify new drug targets. Our hypothesis is that diabetic conditions and AngII coordinately re-program the VSMC transcriptome and epigenome, which lead to persistent dysregulation of genes promoting Phe-sw to unique cellular states underlying VSMC dysfunction and accelerated DCVD. This hypothesis is supported by extensive new preliminary data that show: i) a diabetic state augments AngII actions and promotes VSMC proliferation and Phe-sw gene profiles, which persist even after glucose normalization; ii) DNA methylation is decreased, and chromatin accessibility is increased at key upregulated ECM and inflammatory genes in VSMCs from diabetic mice, even after culture in normal glucose; iii) new cell clusters indicative of Phe-sw occur in aortas of Ang II-infused mice, identified using integrated scRNA- and scATAC-seq; iv) diabetic stimuli induce 3D chromatin changes in vascular cells, seen using chromatin conformation assays. We will test our hypothesis in 3 specific aims: 1) Define diabetes-induced transcriptomic and epigenomic changes [AngII-(in)dependent] in VSMCs at Phe-sw-related genes, and the memory of their persistent dysregulation after glucose normalization in vitro; 2) Elucidate the de-differentiated VSMC subtypes and their functions in diabetes- and AngII-induced Phe-sw in arteries, and their persistence after glucose normalization in vivo, using scRNA-seq and scATAC-seq; and 3) Determine the translational potential for reversing DCVD and vascular memory by targeting candidate genes/loci mediating diabetes- and AngII-induced VSMC Phe-sw. This innovative study, using cutting-edge technologies and functional in vivo models, will provide novel insights into VSMC regulatory networks and epigenetic memory of diabetic vasculopathy. This know...