title: Oscillating glucose induces microRNA-185 and impairs an efficient antioxidant response in human endothelial cells.
abstract: BACKGROUND
  Intracellular antioxidant response to high glucose is mediated by Cu/Mn-superoxide dismutases (SOD-1/SOD-2), catalase (CAT) and glutathione peroxidases (GPx), particularly glutathione peroxidase-1 (GPx-1). Although oscillating glucose can induce a more deleterious effect than high glucose on endothelial cells, the mechanism by which oscillating glucose exerts its dangerous effects is incompletely understood; however, the involvement of oxidative damage has been generally accepted. In this study we sought to determine whether oscillating glucose differentially modulates antioxidant response, and to elucidate the potential regulatory mechanisms exerted by the microRNA-185 (miR-185).
  
  
  METHODS
  Human endothelial cells were exposed for 1 week to constant and oscillating high glucose. SOD-1, SOD-2, CAT and GPx-1, as well as two markers of oxidative stress [8-hydroxy-2'-deoxyguanosine (8-OHdG) and the phosphorylated form of H2AX (γ-H2AX)] were measured at the end of the experiment. Intracellular miR-185 was measured and loss-of function assays were performed in HUVEC. Bioinformatic tool was used to predict the link between miR-185 on 3'UTR of GPx-1 gene. Luciferase assay was performed to confirm the binding on HUVEC.
  
  
  RESULTS
  After exposure to constant high glucose SOD-1 and GPx-1 increased, while in oscillating glucose SOD-1 increased and GPx-1 did not. SOD-2 and CAT remained unchanged under both conditions. A critical involvement of oscillating glucose-induced miR-185 in the dysregulation of endogenous GPx-1 was found. Computational analyses predict GPx-1 as miR-185's target. HUVEC cultures were used to confirm glucose's causal role on the expression of miR-185, its target mRNA and protein and finally the activation of antioxidant response. In vitro luciferase assays confirmed computational predictions targeting of miR-185 on 3'-UTR of GPx-1 mRNA. Knockdown of miR-185, using anti-miR-185 inhibitor, was accompanied by a significant upregulation of GPx-1 in oscillating glucose. 8-OHdG and γ-H2AX increased more in oscillating glucose than in constant high glucose.
  
  
  CONCLUSIONS
  Glucose oscillations may exert more deleterious effects on the endothelium than high glucose, likely due to an impaired response of GPx-1, coupled by the upregulation of miR-185.
authors.affiliation: Department of Cardiovascular Research, IRCCS MultiMedica, Milan, MI, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: La Sala
authors.affiliation: Department of Cardiovascular Research, IRCCS MultiMedica, Milan, MI, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Cattaneo
authors.affiliation: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomedica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain.
authors.forename: Valeria
authors.initials: V
authors.lastname: De Nigris
authors.affiliation: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomedica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Pujadas
authors.affiliation: Experimental Models in Clinical Pathology, INRCA-IRCCS National Institute, Ancona, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Testa
authors.affiliation: Scientific Direction, INRCA, Via S. Margherita, 5, Ancona, 60124, Italy.
authors.forename: Anna R
authors.initials: AR
authors.lastname: Bonfigli
authors.affiliation: Department of Cardiovascular Research, IRCCS MultiMedica, Milan, MI, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Genovese
authors.affiliation: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomedica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain. aceriell@clinic.ub.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ceriello
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism; D000094765:Glutathione Peroxidase GPX1; D000099156:Superoxide Dismutase 2
chemical_list: D000975:Antioxidants; D002374:Catalase; D005947:Glucose; D005979:Glutathione Peroxidase; D035683:MicroRNAs; D013482:Superoxide Dismutase; D000094765:Glutathione Peroxidase GPX1; D000099156:Superoxide Dismutase 2; C552431:MIRN185 microRNA, human; C000722067:GPX1 protein, human
keywords: Antioxidant defense; GPx-1; Oscillating glucose; Oxidative stress; miR-185