title: Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury.
abstract: Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue exert protection against ischemia/reperfusion injury in a distant organ. However, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms within the target organ and the released factors. Here we established a human cell culture model to investigate cellular and molecular effects of RIPC and to identify factors responsible for RIPC-mediated intestinal protection. Human umbilical vein cells (HUVEC) were exposed to repeated episodes of hypoxia (3 × 15 min) and conditioned culture media (CM) were collected after 24h. Human intestinal cells (CaCo-2) were cultured with or without CM and subjected to 90 min of hypoxia/reoxygenation injury. Reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, hydrogen peroxide measurements and lactate dehydrogenase (LDH) assays were performed. In HUVEC cultures hypoxic conditioning did not influence the profile of secreted proteins but led to an increased gelatinase activity (P<0.05) in CM. In CaCo-2 cultures 90 min of hypoxia/reoxygenation resulted in morphological signs of cell damage, increased LDH levels (P<0.001) and elevated levels of hydrogen peroxide (P<0.01). Incubation of CaCo-2 cells with CM reduced the hypoxia-induced signs of cell damage and LDH release (P<0.01) and abrogated the hypoxia-induced increase of hydrogen peroxide. These events were associated with an enhanced phosphorylation status of the prosurvival kinase Erk1/2 (P<0.05) but not Akt and STAT-5. Taken together, CM of hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury. The established culture model may help to unravel RIPC-mediated cellular events and to identify molecules released by RIPC.
authors.affiliation: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Hummitzsch
authors.affiliation: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Zitta
authors.affiliation: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Bein
authors.affiliation: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Steinfath
authors.affiliation: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany. Electronic address: martin.albrecht@uksh.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Albrecht
mesh_terms: D018938:Caco-2 Cells; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism* / Q000494:pharmacology*; D019610:Cytoprotection / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007410:Intestinal Diseases / Q000473:pathology / Q000517:prevention & control; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects* / Q000473:pathology; D019194:Ischemic Preconditioning / Q000379:methods; D010766:Phosphorylation / Q000187:drug effects; D015427:Reperfusion Injury / Q000473:pathology / Q000517:prevention & control*
chemical_list: D017077:Culture Media, Conditioned; D006861:Hydrogen Peroxide
keywords: Endothelial cells; Hypoxia; Intestinal ischemia; Ischemia/reperfusion injury; Remote ischemic preconditioning