title: Advanced glycation end products accelerate rat vascular calcification through RAGE/oxidative stress.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These results suggest that AGEs enhance vascular calcification partly through a RAGE/oxidative stress pathway.
authors.affiliation: Department & Institute of Cardiology, Zhongda Hospital, Southeast University, Nanjing, Jiangsu 210009, PR China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wei
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Ren
authors.forename: Yibo
authors.initials: Y
authors.lastname: Jiang
authors.forename: Hong
authors.initials: H
authors.lastname: Jin
authors.forename: Naifeng
authors.initials: N
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Li
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism*; D001018:Aortic Diseases / Q000139:chemically induced / Q000209:etiology* / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001786:Blood Glucose / Q000378:metabolism; D002478:Cells, Cultured; D002762:Cholecalciferol; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000139:chemically induced / Q000150:complications* / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000097:blood / Q000378:metabolism*; D007328:Insulin / Q000097:blood; D008055:Lipids / Q000097:blood; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D000074624:NADPH Oxidase 1; D009538:Nicotine; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D015398:Signal Transduction; D013311:Streptozocin; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D061205:Vascular Calcification / Q000139:chemically induced / Q000209:etiology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D001786:Blood Glucose; D017127:Glycation End Products, Advanced; D007328:Insulin; D008055:Lipids; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C081628:advanced glycation end products-bovine serum albumin; D013481:Superoxides; D002762:Cholecalciferol; D012710:Serum Albumin, Bovine; D008315:Malondialdehyde; D013311:Streptozocin; D009538:Nicotine; D013482:Superoxide Dismutase; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614712:NOX1 protein, rat; D000469:Alkaline Phosphatase