Tripeptide DT-109 (Gly-Gly-Leu) attenuates atherosclerosis and vascular calcification in nonhuman primates

Abstract Advanced atherosclerotic lesions and vascular calcification substantially increase the risk of cardiovascular events. However, effective strategies for preventing or treating advanced atherosclerosis and calcification are currently lacking. This study investigated the efficacy of DT-109 (Gly-Gly-Leu) in attenuating atherosclerosis and calcification in nonhuman primates, exploring its broader therapeutic potential. In this study, twenty male cynomolgus monkeys were administered a cholesterol-rich diet ad libitum for 10 months. Then, the animals were treated either orally with DT-109 (150 mg/kg/day) or a vehicle (H 2 O) for 5 months while continuing on the same diet. Plasma lipid levels were measured monthly and at the end of the experiment, pathological examinations of the aortas and coronary arteries and RNA sequencing of the coronary arteries were performed. To explore possible molecular mechanisms, the effects of DT-109 on smooth muscle cells (SMCs) were examined in vitro. We found that DT-109 administration significantly suppressed atherosclerotic lesion formation in both the aorta and coronary arteries. Pathological examinations revealed that DT-109 treatment reduced lesional macrophage content and calcification. RNA sequencing analysis showed that DT-109 treatment significantly downregulated the pro-inflammatory factors NLRP3 , AIM2 , and CASP1 , the oxidative stress factors NCF2 and NCF4 , and the osteogenic factors RUNX2 , COL1A1 , MMP2 , and MMP9 , while simultaneously upregulating the expression of the SMCs contraction markers ACTA2 , CNN1 , and TAGLN . Furthermore, DT-109 inhibited SMC calcification and NLRP3 inflammasome activation in vitro. These results demonstrate that DT-109 effectively suppresses both atherosclerosis and calcification. These findings, in conjunction with insights from our previous studies, position DT-109 as a novel multifaceted therapeutic agent for cardiovascular diseases.