journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/ reoxygenation injury Lars Hummitzsch, Karina Zitta, Berthold Bein, Markus Steinfath, Martin Albrecht n Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany articleinformation Article Chronology: Received 10 September 2013 Received in revised form 17 December 2013 Accepted 26 December 2013 Available online 4 January 2014 Keywords: Remote ischemic preconditioning Endothelial cells Hypoxia Intestinal ischemia Ischemia/reperfusion 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 Â 15 min) and conditioned culture media (CM) were collected after 24 h. 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 (Po0.05) in CM. In CaCo-2 cultures 90 min of hypoxia/ reoxygenation resulted in morphological signs of cell damage, increased LDH levels (Po0.001) and elevated levels of hydrogen peroxide (Po0.01). Incubation of CaCo-2 cells with CM reduced the hypoxia-induced signs of cell damage and LDH release (Po0.01) and abrogated the hypoxia- induced increase of hydrogen peroxide. These events were associated with an enhanced phosphorylation status of the prosurvival kinase Erk1/2 (Po0.05) 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. & 2014 Elsevier Inc. All rights reserved. Introduction Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue induce protection against ischemia/reperfusion (I/R) injury in a distant organ. This powerful mechanism of organ protection can be simply induced by transient occlusion of blood flow to a limb with a blood pressure cuff [1]. Over the last years several clinical and 0014-4827/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.12.022 n Corresponding author. Fax: þ49 431 597 3704. E-mail address: martin.albrecht@uksh.de (M. Albrecht). EXPERIMENTAL CELL RESEARCH 322 (2014) 62 – 70