Abstract Body: The diabetic state of chronic hyperglycemia and systemic inflammation alters endothelial cell (EC) phenotype, promoting endothelial-to-mesenchymal cell transition (EndMT) and pathological vascular remodeling. EndMT skews ECs towards mesenchymal structural cells with impaired integrity. The epigenetic and molecular mechanisms of this process are not fully known, providing an opportunity to expand our understanding of diabetes-induced vascular disease. NAD⁺-dependent histone deacetylases (Sirtuins) are reported to control EC function and anti-inflammatory cytokine transcription. We hypothesized that diabetes promotes vascular dysfunction through macrophage-EC interactions that lead to pathological EndMT, mediated in part by Sirtuin activity. To assess this, we first performed vascular physiologic studies on db/db mice compared to db/het littermates (N = 6/group). These included pulse-wave velocity (PWV), wire myography, and GlycoCheck™ of their aortic and mesenteric vessels. Bone marrow derived macrophages (BMDMs) and primary pulmonary ECs were isolated. Next, non-diabetic ECs were exposed to diabetic BMDM-conditioned media (± recombinant SIRT1) and analyzed for EndMT markers. Physiologically, diabetic mice showed trends toward increased arterial stiffness (PWV: 2.56±0.06 vs 3.38±0.06 m/s), impaired glycocalyx integrity (PBR: 1.245±0.016 vs 1.283±0.021 µm), and reduced endothelium-dependent vasodilation (87.3±3.7% vs 79.5±1.7%), with preserved smooth-muscle function (66.7±7.7% vs 67.6±4.9%). BMDMs from diabetic mice expressed more pro-EndMT cytokines Il6 (1.00±0.03 vs 1.23±0.04) and Tgfb1 (1.00±0.04 vs 1.23±0.02). At baseline, ECs from diabetic mice showed reduced Sirt1 (1.00±0.01 vs 0.487±0.005), Sirt3 (1.00±0.03 vs 0.845±0.052), Sirt6 (1.00±0.03 vs 0.747±0.088), and nicotinamide phosphoribosyltransferase Nampt (1.00±0.03 vs 0.411±0.041), which controls upstream NAD⁺ production (and thus, Sirtuin expression). Molecularly, when exposed to diabetic BMDM-conditioned media, non-diabetic ECs showed impaired cellular morphology and increased expression of mesenchymal markers Acta2 (0.939±0.145 vs 1.962±0.305) and S100a4 (0.799±0.028 vs 1.104±0.033), suggesting a myofibrotic predisposition. These effects were reversed with rSIRT1 treatment (Acta2 0.877±0.089; S100a4 0.854±0.072), supporting a therapeutic role for SIRT1 in limiting diabetes-associated vascular remodeling. Moving forward, we will evaluate SiRT1 in diabetes-mediated prevention of aortic aneurysms.