title: CML/RAGE signal induces calcification cascade in diabetes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Zhongqun
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Pathology, Affiliated Hospital of Jiangsu University, Zhenjiang, 212001 China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Ultrasound, Affiliated Hospital of Jiangsu University, Zhenjiang, 212001 China.
authors.forename: Rui
authors.initials: R
authors.lastname: Du
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Jinchuan
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department and Institute of Cardiology, Zhongda Hospital, Southeast University, 87 Dingjiaqiao, Nanjing, 210009 China.
authors.forename: Naifeng
authors.initials: N
authors.lastname: Liu
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yuan
authors.affiliation: Department of Cardiology, Huaian No.1 People's Hospital, Huaian, 223300 China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Suining
authors.initials: S
authors.lastname: Xu
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.affiliation: Department of Endocrinology, Affiliated Hospital of Jiangsu University, Zhenjiang, 212001 China.
authors.forename: Guoyue
authors.initials: G
authors.lastname: Yuan
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Baohai
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, 438 Jiefang, Zhenjiang, 212001 China.
authors.forename: Peijing
authors.initials: P
authors.lastname: Liu
keywords: Advanced glycation end-products; Atherosclerosis; Diabetic foot; Vascular calcification; p38MAPK