Advanced glycation end-product Nε-carboxymethyl-Lysine accelerates progression of atherosclerotic calcification in diabetes.

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(ε)-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(-/-) 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 (10mg/kg/day). The mice were euthanized and analyzed at 0 month (group 0M, n = 10), 2 months (group 2M, n = 10), and 4 months (group 4M, 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 μg/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 μg/mL of RAW264.7-derived-apoptotic bodies and 50 μg/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 μmol/L CML plus 50 μg/mL oxLDL, and 80 μg/mL apoptotic bodies (ABs) for 7 days, semi-quantitative analysis of bone morphogenetic protein 2 (BMP-2), core-binding factor α1 (cbfα1), and alkaline phosphatase (ALP) expression showed 5.0-, 2.0-, and 2.9-fold increases, respectively, compared with those in 50 μg/mL oxLDL and 80 μg/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-cbfα1-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.