CML/RAGE signal induces calcification cascade in diabetes

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 cal...

Mechanisms of Matrix Vesicles Mediating Calcification Transition in Diabetic Plaque

“…Our previous study found that Ne-carboxymethyl-lysine (CML), a key component of serum AGEs, can be seen as an early indicator for the calcification of anterior tibial artery plaque in patients with diabetic amputation [29]. Further, our research group successfully replicated the animal and cell model of calcification in diabetic plaque using CML [30], finding that the calcification of the anterior tibial artery plaque in patients with diabetic foot amputation showed the appearance of focal microcalcification [31]. In addition, our research group previously synthesised the CML fluorine-18 ( 18 F)-labelled tracer, and carried out a pharmacokinetic study of the novel molecular probe under physiological and pathological conditions [32].…”

“…It is known that AGEs have two major receptors in atherosclerotic plaque: receptor for advanced glycation end products (RAGE) and galectin-3 (Gal-3) [27,31,37,38]. Mainly distributed in microcalcifications and unstable plaques caused by inflammation, RAGE, a membrane receptor with a transmembrane domain, is mostly found in inflammatory infiltrating mononuclear macrophages and SMCs [39].…”

“…Mainly distributed in microcalcifications and unstable plaques caused by inflammation, RAGE, a membrane receptor with a transmembrane domain, is mostly found in inflammatory infiltrating mononuclear macrophages and SMCs [39]. Differently from the distribution of RAGE, Gal-3 is mainly expressed in macrocalcification, non-inflammatory plaques, and areas of VSMC fibrosis, which can be distributed in the circulation, extracellular matrix and nucleus, plasma, and membranes, but it lacks in transmembrane domain and is rarely seen in areas of inflammatory vulnerable plaques and microcalcifications [9,31,37,38]. In vitro studies have shown that blocking Gal-3 is associated with a decrease in the diameter of SMC extracellular matrix calcified nodules and an increase in the number of nodules [24].…”

“…What is known is that AGEs bind to their intratissue receptors and mediate the evolution of diabetic vascular calcification mainly through transforming growth factor (TGF)-b, mitogen-activated protein kinase (MAPK), Akt, and nuclear factor kappa B (NF-kB) pathways [50][51][52][53]. Our previous studies suggested that the AGE CML binds to RAGE and mediates microcalcification in diabetic atherosclerotic plaque via the p38/MAPK pathway [31]. Does RAGE/Gal-3 induce changes in the scaffolding effector molecule of sortilin and its protein complex via the TGF-b, MAPK, Akt, and NF-kB pathways, leading to the release and enrichment of MV, as well as transition of micro-and macrocalcification?…”

Role of the Enterocyte in Fructose-Induced Hypertriglyceridaemia

“…Although evidence concerning the effects of fructose-derived AGEs remains limited, it has recently been posited that they may exacerbate hepatic lipogenesis via inactivation of hepatic adenosine monophosphate-activated protein kinase [48]. A thorough discussion of the effects of MG and fructose-derived AGEs is beyond the scope of the current review, however, and readers are referred to several recent articles on the effects and potential mechanisms of AGEs [47,49,50,51,52]. …”