Advanced glycation end products accelerate rat vascular calcification through RAGE/oxidative stress

BMC Cardiovascular Disorders (Impact Factor: 1.88). 03/2013; 13(1):13. DOI: 10.1186/1471-2261-13-13
Source: PubMed


Arterial media calcification (AMC) is highly prevalent and is a major cause of morbidity, mortality, stroke and amputation in patients with diabetes mellitus (DM). Previous research suggests that advanced glycation end products (AGEs) are responsible for vascular calcification in diabetic patients. The potential link between oxidative stress and AGEs-induced vascular calcification, however, has not been examined.

Male Wistar rats received a high fat diet for 8 weeks followed by a single dose of streptozotocin to induce DM (DM). Calcification was induced with Vitamin D3 and nicotine (VDN). We started VDN treatment at 1 week after the initial streptozotocin injection (DM+VDN). Age-matched rats were used as controls (CON). Metabolic parameters, aortic calcium content, alkaline phosphatase (ALP) protein, malondialdehyde (MDA) content, Cu/Zn superoxide dismutase (SOD) activity, aorta receptor for advanced glycation end products (RAGE) and aorta AGEs levels were measured. In vitro, vascular smooth muscle cells (VSMCs) were cultured with AGEs in DMEM containing 10 mmol·L-1 ß -glycerophosphate (ß-GP). Calcium content and ALP activity were used to identify osteoblastic differentiation and mineralization. Western blots were used to examine protein expression of Cu/Zn SOD, NADPH oxidase Nox1 and RAGE. In addition, the intracellular reactive oxygen species (ROS) generation was evaluated using fluorescent techniques with dihydroethidine (DHE) method.

The DM+VDN group showed a significant increase in aortic calcium content, levels of aorta AGEs, MDA content, ALP protein levels and RAGE expression, although Cu/Zn SOD activity decreased significantly. In vitro, enhanced Nox1, RAGE expression as well as the production of intracellular superoxide anions, and reduced expression of Cu/Zn SOD induced by AGEs were attenuated by the anti-RAGE antibody or a ROS inhibitor. Furthermore, the AGEs-stimulated ROS increase was also significantly inhibited by a SOD mimetic. Increased ALP activity and calcium deposition were also inhibited markedly by the ROS inhibitor and the anti-RAGE antibody.

These results suggest that AGEs enhance vascular calcification partly through a RAGE/oxidative stress pathway.

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    • "Advanced glycation end products (AGEs) derived from reducing sugars reaction non-enzymatically constitute a diverse group of compounds formed when glucose or other reducing sugars such as galactose and fructose react with amino acids, nucleotide bases or fatty acids, forming glycated molecules [4]. AGEs play an important role in the pathogenesis of numerous diseases, including diabetic complications, aging and atherosclerosis [5] [6]. "
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    ABSTRACT: Diabetic nephropathy as the most common cause of end-stage renal disease accounts for a significant increase in morbidity and mortality in patients. Epithelial to mesenchymal transition (EMT) of tubular cells is associated with diabetic nephropathy. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of diabetic nephropathy via multifactorial mechanisms.However,whether AGEs could induce EMT in Tubular epithelial cells is still unkown. In this study,we found that AGEs induced EMT and accompanied by reduced expression of the epithelial markers E-cadherin and enhanced expression of the mesenchymal markers vimentin and alpha-smooth muscle actin. Furthermore, the expression of HMGA2 was upregulated by AGEs. Far more interesting, its knockdown by short interfering RNA (siRNA) effectively reversed AGEs-induced EMT. Meanwhile, we also found that knockdown of HMGA2 inhibited high AGEs-induced generation of reactive oxygen species (ROS) and the activation of p38 MAPK. Collectively, these studies suggest that HMGA2 plays a important role in EMT during Diabetic nephropathy and more study toward HMGA2 should be played in renal pathogenesis. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 01/2015; 457(4). DOI:10.1016/j.bbrc.2015.01.063 · 2.30 Impact Factor
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    • "Therefore, other mechanisms besides LGI and ED might be involved in the association of AGEs with CAC. Recent publications show that AGEs-induced vascular calcification in rat vascular smooth muscle cells (VSMCs) is mediated by oxidative stress in vitro [44,45], and oxidative stress may thus provide an additional mechanism explaining the association of AGEs with CAC. "
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    ABSTRACT: Advanced glycation endproducts (AGEs) may play a role in the development of coronary artery calcification (CAC) in type 1 diabetes (T1DM). We studied plasma AGEs in association with T1DM and CAC, and whether or not the latter association could be explained by low-grade inflammation (LGI) or endothelial dysfunction (ED). We studied 165 individuals with and 169 without T1DM. CAC was quantified in a CAC score based on CT-scanning. Plasma levels of protein-bound pentosidine, Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL) were measured with HPLC/UPLC with fluorescence detection or tandem-mass spectrometry. Tetrahydropyrimidine (THP) was measured with ELISA, as were HsCRP, and sVCAM-1 and vWF, as markers for LGI and ED, respectively. Associations were analyzed with ANCOVA and adjusted for age, sex, BMI, waist-to-hip ratio, smoking, blood pressure, lipid profile, eGFR and T1DM. Individuals with T1DM had higher plasma levels of pentosidine, CML and THP compared with controls; means (95%CI) were 0.69 (0.65-0.73) vs. 0.51 (0.48-0.54) nmol/mmol LYS, p < 0.001; 105 (102--107) vs. 93 (90--95) nmol/mmol LYS, p < 0.001; and 126 (118--134) vs. 113 (106--120) U/mL, p = 0.03, respectively. Levels of pentosidine were higher in individuals with T1DM with a moderate to high compared with a low CAC score, means (95%CI) were 0.81 (0.70-0.93) vs. 0.67 (0.63-0.71) nmol/mmol LYS, p = 0.03, respectively. This difference was not attenuated by adjustment for LGI or ED. We found a positive association between pentosidine and CAC in T1DM. These results may indicate that AGEs are possibly involved in the development of CAC in individuals with T1DM.
    Cardiovascular Diabetology 10/2013; 12(1):149. DOI:10.1186/1475-2840-12-149 · 4.02 Impact Factor
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    • "Among the various oxidative modifications of proteins, carbonylation is a critical and irreparable modification that has been shown to be elevated during aging, diabetes and in neurodegeneration [12], [13], [14], [15], [16], [17], [18]. Diabetic subjects are known to have elevated glucose-, and lipoxidation-derived carbonyl stress [18], [19], [20], [21], [22], [23], however, it remains unknown if carbonylation of sciatic nerve/myelin proteins are elevated in peripheral neuropathies. Therefore, we asked (i) does oxidative stress have an effect on carbonylation and misfolding of sciatic nerve proteins in peripheral neuropathy, and (ii) is the carbonylation of sciatic nerve/myelin proteins associated with loss in nerve conduction and myelin in peripheral neuropathy? "
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    ABSTRACT: Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1(-/-) ) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.
    PLoS ONE 06/2013; 8(6):e65725. DOI:10.1371/journal.pone.0065725 · 3.23 Impact Factor
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