title: Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis.
abstract: OBJECTIVE
  Oxysterol activity on the erythrocyte (RBC) programmed cell death (eryptosis) had not been studied yet. Effects of an oxysterol mixture in hyper-cholesterolemic-relevant proportion, and of individual compounds, were investigated on RBCs from healthy humans.
  
  
  METHODS
  Membrane phosphatidylserine (PS) externalization, calcium entry, ROS production, amino-phospholipid translocase (APLT) activity were evaluated by cytofluorimetric assays, cell volume from forward scatter. Prostaglandin PGE2 was measured by ELISA; GSH-adducts and lipoperoxides by spectrophotometry. Involvement of protein kinase C and caspase was investigated by inhibitors staurosporin, calphostin C, and Z-DEVD-FMK, respectively.
  
  
  RESULTS
  Oxysterols caused PS externalization and cell shrinkage, associated with PGE2release, opening of PGE2-dependent calcium channels, ROS production, GSH depletion, membrane lipid oxidation. Addition of antioxidants prevented Ca(2+) influx and eryptosis. Calcium removal prevented cell shrinkage, with small effect (-20%) on the PS exposure, whereas ROS generation was unaltered. Either in the presence or absence of calcium i) oxysterols inhibited APLT, ii) staurosporin, calphostin C, Z-DEVD-FMK blunted and iii) antioxidants fully prevented the oxysterol-induced PS externalization. Only 7-ketocholesterol and cholestan-3β,5α,6β-triol were individually active. Eryptosis was observed in RBCs isolated after ex vivo spiking of human whole blood with the oxysterol mixture.
  
  
  CONCLUSIONS
  Oxysterols induce an oxidative stress-dependent eryptosis, involving calcium-independent mechanisms. Eryptotic activity of oxysterols may be relevant in vivo.
authors.affiliation: Department of Biological Chemical and Pharmaceutical Science and Technologies (STEBICEF), Universita di Palermo, Palermo, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Tesoriere
authors.forename: Alessandro
authors.initials: A
authors.lastname: Attanzio
authors.forename: Mario
authors.initials: M
authors.lastname: Allegra
authors.forename: Antonio
authors.initials: A
authors.lastname: Cilla
authors.forename: Carla
authors.initials: C
authors.lastname: Gentile
authors.forename: Maria A
authors.initials: MA
authors.lastname: Livrea
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D006937:Hypercholesterolemia / Q000378:metabolism / Q000473:pathology*; D007653:Ketocholesterols / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; D015220:Calcium Channels; D007653:Ketocholesterols; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D011493:Protein Kinase C; D020169:Caspases; D015232:Dinoprostone; C003001:7-ketocholesterol; D002118:Calcium