Advanced glycation end-product N^蓻-carboxymethyl-Lysine accelerates progression of atherosclerotic calcification in diabetes

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• 作者：Zhongqun Wang^a ; Yicheng Jiang^b ; Naifeng Liu^a ; ^{liunf@seu.edu.cn} ; Liqun Ren^a ; Yinghong Zhu^a ; Yanli An^c ; Dengyu Chen^d
• 关键词：Advanced glycation end-products ; Apoptosis ; Phenotype transformation ; Vascular smooth muscle cells ; Vascular calcification ; Atherosclerosis ; Diabetes
• 刊名：Atherosclerosis
• 出版年：2012
• 期刊代码：28_00219150
• 类别：med
• 出版时间：April, 2012
• 卷：221
• 期：2
• 页码：387-396
• 文件大小：1689 K

摘要

Objective

Vascular calcification is an active deposition process of calcium phosphate which resembles bone formation and is highly regulated by osteoblast-like cells. Existing studies demonstrate that advanced glycation end-products (AGEs) may play a pathogenic role in the vascular calcification process. However, their mechanism remains poorly understood. The aim of our current study is to investigate how non-cross-link and non-fluorescent N^蓻-carboxymethyl-Lysine (CML), a major immunogen of AGEs, affect the progression of atherosclerotic calcification in diabetes.

Methods

The present study consisted of an in vivo investigation and two in vitro investigations. In study I, male apoE^{鈭?鈭?/sup> mice were first rendered diabetic by the administration of 5 daily intraperitoneal injections of streptozotocin (STZ, 40 mg/kg), and then given a semi-synthetic high-fat diet (HFD) plus daily injections of CML (10 mg/kg/day). The mice were euthanized and analyzed at 0 month (group 0 M, n = 10), 2 months (group 2 M, n = 10), and 4 months (group 4 M, n = 10) after the triple administrations of STZ-CML-HFD. In study II, the effects of CML on the apoptosis in macrophages were investigated. RAW264.7 cells were incubated with or without 50 渭g/mL oxLDL plus various concentrations of CML for 48 h. In study III, we investigated whether A7r5 aortic smooth muscle cells were induced into osteoblast-like phenotypes by incubation with or without 80 渭g/mL of RAW264.7-derived-apoptotic bodies and 50 渭g/mL of oxLDL plus various concentrations of CML (or high-glucose) for 7 days. Related analyses (i.e., H&E staining, Masson staining, von Kossa staining, TUNEL staining, immunohistochemical staining, calcium content assay, annexin V-FITC/PI double-staining, and Western blot) were performed.}

Results

Morphological analysis showed that early atherosclerotic plaques appeared 2 months after the triple administrations of STZ-CML-HFD, and that typically advanced plaques with extensive calcification lesions, abundant cholesterol crystals, and proliferative collagen were formed 4 months after the triple administrations of STZ-CML-HFD. Furthermore, CML deposition signals and the expression of receptor for advanced glycation end-products (RAGE) in the aortic wall were mainly restricted in the atherosclerotic plaques. After the incubation of A7r5 smooth muscle cells with 10 渭mol/L CML plus 50 渭g/mL oxLDL, and 80 渭g/mL apoptotic bodies (ABs) for 7 days, semi-quantitative analysis of bone morphogenetic protein 2 (BMP-2), core-binding factor 伪1 (cbf伪1), and alkaline phosphatase (ALP) expression showed 5.0-, 2.0-, and 2.9-fold increases, respectively, compared with those in 50 渭g/mL oxLDL and 80 渭g/mL ABs. Subsequently, a similar trend was observed in the calcium deposition of the cell layer. However, high-glucose had no effects on the ALP activity and calcium deposition of A7r5 cell layer under high-lipid, apoptosis-coexisting conditions. Both animal and cell studies consistently demonstrated that the CML/RAGE axis may first initiate the apoptosis of macrophages in atherosclerotic lesions and then induce BMP-2-cbf伪1-ALP-calcification cascade in a high-lipid, apoptosis-coexisting environment.

Conclusion

The CML/RAGE axis may play an important role in atherosclerotic calcification of diabetes through the mechanism that induces the apoptosis of macrophages followed by the osteogenic differentiation of aortic smooth muscle cells.