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 (H₂O) 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.

Fig. 1

DT-109 ameliorates atherosclerotic lesions and macrophage infiltration. a Schematic diagram of the experimental approach. Twenty cynomolgus monkeys (male, age ≥ 9 years, BMI > 30) were fed a high cholesterol diet (HCD) for 10 months and then randomly divided to receive either DT-109 (150 mg/kg/d) or vehicle (H₂O) for an additional 5 months, followed by pathological analysis of the aorta. b Lipid levels during 5 months of DT-109 treatment. Levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were measured (n = 10 for each group). Unpaired comparisons between vehicle and DT-109 groups at each time point were performed using the Kruskal-Wallis test; * p < 0.05. c En face monkey aorta staining with Sudan IV (vehicle, n = 9; DT-109, n = 10). d Statistical analysis of the aortic lesions and the percentage of plaque in each part of the aorta. e Histological staining of the aortic arch with hematoxylin and eosin (H&E), CD68 (macrophage marker), ACTA2 (actin alpha 2, smooth muscle cell marker, SMC), and Masson’s trichrome staining. Scale bar: 200 μm. f Statistical analysis of the aortic arch intimal lesions, CD68, ACTA2, and Masson staining areas (vehicle, n = 9; DT-109, n = 10). Data are presented as mean ± SD. All statistical comparisons between groups were evaluated by Kruskal-Wallis test. The exact p value is specified

Fig. 2

DT-109 treatment alleviates atherosclerosis in the coronary arteries. a Representative images of circumflex artery H&E staining, CD68 (for macrophage, MΦ), ACTA2 (for SMC), and Masson’s trichrome staining (vehicle, n = 6; DT-109, n = 6). Scale bar: 200 μm. b Statistical analysis of coronary stenosis rates and MΦ, SMC, Masson’s trichrome staining positive areas. Data are presented as mean ± SD. All statistical comparisons between groups were evaluated by Kruskal-Wallis test. The exact p value is specified

Fig. 3

DT-109 attenuates vascular calcification in cynomolgus monkeys. a Hematoxylin and eosin (H&E) and calcification staining (Von Kossa) of the aortic arch (scale bar: 100 μm). b Quantification of calcified area in the aortic arch (vehicle, n = 9; DT-109, n = 10). c Extraction of total RNA from monkey common carotid arterial tissue and detection of runx family transcription factor 2 (RUNX2), SRY-box transcription factor 9 (SOX9), cytochrome p450 oxidoreductase (POR) mRNA expression by real-time PCR (n = 5 for each group). Data are presented as mean ± SD. All statistical comparisons between groups were evaluated by Kruskal–Wallis test

Fig. 4

Transcriptomics reveals that DT-109 induces vascular smooth muscle contraction and inhibits inflammation.RNA-sequencing was performed on the right coronary artery from cynomolgus monkeys after treatment with vehicle or DT-109 for 5 months (n = 4 for each group). a Principal components analysis (PCA) of RNA-sequencing data. b Volcano plots of differentially expressed genes (DEGs) (blue: down-regulated; red: upregulated. log2 fold change ≥ 1.5, Q value ≤ 0.05). c, d The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was enriched for DEGs up-regulated and down-regulated in red and blue, respectively. e Gene set enrichment analysis (GSEA) shows enrichment in the vascular smooth muscle contraction and NOD-like receptor signaling pathway. f Gene ontology (GO) enrichment analysis for DEGs, focusing on cellular components (GO-CC) and biological processes (GO-BP). g Heat map of coronary-related DEGs involved in inflammatory and oxidative stress responses

Fig. 5

DT-109 inhibits inflammation and oxidative stress.a Coronary arteries were collected and analyzed for inflammation-associated genes. Real-time PCR analysis of NLR family pyrin domain containing 3 (NLRP3), apoptosis-related cysteine peptidase (CASP1), interleukin 18 (IL-18), and IL-1β expressions in the vehicle and DT-109 treated coronary arteries (normalized to 18S; n = 5 for each group). b, c Upon 24 h of ox-LDL induction, protein levels of NLRP3, absent in melanoma 2 (AIM2), PYD and CARD domain containing (ASC), CASP1, and IL-1β were determined by Western blot in A7r5 cells after vehicle or DT-109 treatment (n = 4 for each group). d A7r5 cells were divided into a control group and an ox-LDL-induced group, treated with or without DT-109 (0.02 mM, 0.1 mM, 0.5 mM), and labeled with DCFH-DA to assess superoxide levels (scale bar: 200 μm). e After ox-LDL stimulation, supernatants from A7r5 cells incubated with or without DT-109 (0.5 mM) were collected to analyze glutathione (GSH) and malondialdehyde (MDA) levels (n = 4 for each group). f Detection of GSH and MDA in carotid artery tissue (n = 5 for each group). Data are presented as mean ± SD. Statistical differences were compared using one-way ANOVA with Tukey’s post hoc analysis or Dunn’s test, multiple comparison adjusted p-values were reported

Fig. 6

DT-109 alleviates oxidized low-density lipoprotein-induced smooth muscle cell osteogenic differentiation in vitro.a Heatmap comparing calcification-related gene expression levels in cynomolgus monkey coronary arteries between vehicle and DT-109 treatment (n = 4 for each group). b Real-time PCR validation of smooth muscle contraction-related transcription factors actin alpha 2, smooth muscle (ACTA2) and calponin 1 (CNN1) normalized with 18S in right coronary arteries isolated from vehicle- and DT-109-treated monkeys (n = 5 for each group). c Real-time PCR validation of vascular calcification-related transcription factors runx family transcription factor 2 (RUNX2), cytochrome p450 oxidoreductase (POR), matrix metallopeptidase 2 (MMP2), and MMP9 normalized with 18S in monkey coronary arteries (n = 5 for each group). d Effect of DT-109 on smooth muscle contraction markers (ACTA2 and CNN1) in HASMC and A7r5 cells stimulated with or without ox-LDL for 24 h (n = 4 biologically independent samples). e Effect of DT-109 on osteogenic differentiation markers (RUNX2 and POR) in A7r5 cells stimulated with or without ox-LDL for 24 h (n = 4 for each group). Data are presented as mean ± SD; all data points are shown. Statistical differences were compared using one-way ANOVA with Tukey’s post hoc analysis or Dunn’s test, multiple comparison adjusted p-values were reported

Fig. 7

DT-109 inhibits smooth muscle cell osteogenic differentiation.a Alizarin red stained images of A7r5 in the vehicle- or DT-109 (0.5 mM and 1 mM)-treated group in response to calcified media (CM) induced for 7 d (Scale bar: 500 μm). b Detection of Ca²⁺ concentration in A7r5 cell suspensions after DT-109 (0.5 mM and 1 mM) treatment (n = 5). c Protein levels of NLR family pyrin domain containing 3 (NLRP3), PYD and CARD domain containing (ASC), apoptosis-related cysteine peptidase (CASP1), and interleukin 1 beta (IL-1β) in A7r5 cells incubated with CM induced for 7 d or basal medium with or without DT-109 (1 mM). d Quantitative analysis of protein levels (n = 4). e Protein levels of runx family transcription factor 2 (RUNX2) and collagen type I alpha 1 chain (COL1A1) in A7r5 cells incubated in CM for 7 d or basal medium with or without DT-109 (1 mM). f Quantitative analysis of protein levels (n = 4). g A schematic illustrating the potential mechanisms by which DT-109 influences atherosclerosis. Online painting: https://app.biorender.com/. Data represent the mean ± SD analyzed by one-way ANOVA with Tukey’s post hoc analysis or Dunn’s test, multiple comparison adjusted p-values were reported