Abstract
ObjectiveVascular 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.MethodsIn 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.ResultsMorphological 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.ConclusionSpotty calcification was predominant in the atherosclerotic plaques of amputated diabetic patients. CML/RAGE signal may induce the calcification cascade in diabetes via ROS-p38MAPK.
Highlights
The International Diabetes Federation reported that the number of diabetic patients worldwide was 387 million in 2014 and is predicted to reach 592 million by 2035 [1]
Spotty calcification was predominant in the atherosclerotic plaques of diabetic patients with amputation, and macrocalcification was almost invisible
Semi-quantitative Western blot analysis suggested that the intensity of CML/receptor for advanced glycation end products (RAGE) signal increased with progression of atherosclerotic calcification in diabetic patients
Summary
The International Diabetes Federation reported that the number of diabetic patients worldwide was 387 million in 2014 and is predicted to reach 592 million by 2035 [1]. China has the highest number of people with diabetes, which accounts for about one-third of the total number of diabetic patients worldwide [1]. Patients with diabetes commonly manifest vascular calcification, including intima and medial calcification, which are mainly distributed in the coronary artery and lower extremity vessels, respectively [2, 3]. Studies showed that the degree of vascular calcification could be the optimal predictor of cardiovascular mortality, stroke incidence, and risk of lower limb amputation in patients with diabetes mellitus [4,5,6,7,8]. Further insights into the mechanism of calcification in diabetic atherosclerosis exhibit theoretical and social significance
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