Activated endothelial cells (ECs) play an important role in the development of atherosclerosis (AS) because they form neointima resulting from abnormal vascular smooth muscle cell (VSMC) hyperplasia. TET2 was recently reported as a major regulator of the phenotypic switch of VSMCs, and it also protects ECs from noxious stimuli and represses inflammation in AS. The purpose of the present study is to determine whether activation of CD137 signaling can regulate the function of VSMCs by altering endothelial TET2 expression and to explore the potential mechanisms. VSMCs were isolated from C57BL/6 J mice, and the influence of ECs on VSMCs was analyzed using a co-culture system. Western blotting was used to detect the protein expression levels of TET2 and CD9, CD81, and CD63 as well as VSMC phenotypic markers. RT-PCR was performed to detect the TET2 mRNA levels. EdU proliferation assay and transwell migration assay were employed to assess the functions of VSMCs. EXO-spin kits were used to extract and purify exosomes. Transmission electron microscopy and nanoparticle tracking analysis were applied to characterize exosomes. ECs transduced with the TET2-overexpression lentivirus showed that the activation of endothelial CD137 signaling decreased TET2 expression and the TET2 content in exosomes. EC-derived exosomes were internalized by VSMCs, which inhibited phenotypic switching in vitro and neointimal formation in vivo. However, exosomes derived from CD137-activated ECs exhibited weakened protective effects on VSMCs. In conclusion, activation of endothelial CD137 signaling attenuated the repressive effects of EC-derived exosomes on platelet-derived growth factor (PDGF)-BB-induced phenotypic switching of VSMCs and neointimal formation after carotid injury.

Two major issues are involved in clinical atherosclerosis treatment. First, there are no significant clinical markers for early diagnosis of atherosclerosis. Second, the plaque will not regress once it initiates even if the risk factors are removed. In this paper, the research shows that the hypermethylation level of the microRNA 145 (miR-145) promoter is related to a DNMT1 and TET2 dynamic imbalance. The reduction of miR-145 causes NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation through CD137/NFATc1 signaling. These findings could be a potential target for plaque regression in the future.

Vascular calcification is a significant predictor of coronary heart disease events, stroke, and lower-limb amputation. Advanced glycation end-products (AGEs) play a key role in the development of vascular calcification. However, the role of Nε-carboxymethyl-lysine (CML), a major active ingredient of heterogeneous AGEs, in the development of atherosclerotic calcification in diabetic patients and the underlying mechanism remain unclear. Hence, the role and the mechanism of CML in the transmission pathway of diabetic calcification cascade were investigated in the present study. In vivo and in vitro investigations were performed. In study I, 45 diabetic patients hospitalized for above-knee amputation in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were recruited from February 2010 to June 2015. The patients were categorized based on the severity of anterior tibial artery stenosis, which was assessed by color Doppler ultrasound, into mild stenosis (0% < stenosis < 50%, n = 15), moderate stenosis (50 ≤ stenosis < 70%, n = 15), and severe stenosis/occlusion groups (70 ≤ stenosis ≤ 100%, n = 15). In study II, the specific mechanism of CML in the transmission pathway of the diabetic calcification cascade signal was investigated in A7r5 aortic smooth muscle cells under high-lipid, apoptosis-coexisting conditions. ELISA (for serum CML concentration of patients), ultrasound (for plaque size, calcification, blood flow filling, vascular stenosis etc.), H&E staining (for plaque morphology), vonKossa staining (for qualitative analysis of calcification), calcium content assay (for quantitative analysis of calcification), and Western blot analyses of CML, receptor for advanced glycation end products (RAGE), NADPH oxidase 4, phosphorylated p38, core-binding factor α1 (cbfα1), alkaline phosphatase (ALP) and β-actin were then performed. Morphological analysis revealed extensive calcification lesions in the intima and media of the anterior tibial artery. The extent and area of calcium deposition in the intima significantly increased with disease progression. Interestingly, spotty calcification was predominant in the atherosclerotic plaques of diabetic patients with amputation, and macrocalcification was almost invisible. Pearson correlation analysis revealed that serum CML level exhibited a significant positive correlation with calcium content in the arterial wall (R2 = 0.6141, P < 0.0001). Semi-quantitative Western blot analysis suggested that the intensity of CML/RAGE signal increased with progression of atherosclerotic calcification in diabetic patients. In subsequent in vitro study, the related pathway was blocked by anti-RAGE antibody, NADPH oxidase inhibitor DPI, p38MAPK inhibitor SB203580, and anti-cbfa1 antibody in a step-wise manner to observe changes in calcium deposition and molecular signals. Results suggested that CML may play a key role in atherosclerotic calcification mainly through the CML/RAGE- reactive oxygen species (ROS)-p38MAPK-cbfα1-ALP pathway. Spotty calcification was predominant in the atherosclerotic plaques of amputated diabetic patients. CML/RAGE signal may induce the calcification cascade in diabetes via ROS-p38MAPK.