Serum from patients undergoing remote ischemic preconditioning protects cultured human intestinal cells from hypoxia-induced damage: involvement of matrixmetalloproteinase-2 and -9.
Remote ischemic preconditioning (RIPC) can be induced by transient occlusion of blood flow to a limb with a blood pressure cuff and exerts multiorgan protection from ischemia/reperfusion injury. Ischemia/reperfusion injury in the intestinal tract leads to intestinal barrier dysfunction and can result in multiple organ failure. Here we used an intestinal cell line (CaCo-2) to evaluate the effects of RIPC-conditioned patient sera on hypoxia-induced cell damage in vitro and to identify serum factors that mediate RIPC effects. Patient sera (n = 10) derived before RIPC (T0), directly after RIPC (T1) and 1 h after RIPC (T2) were added to the culture medium at the onset of hypoxia until 48 h after hypoxia. Reverse transcription-polymerase chain reaction, lactate dehydrogenase (LDH) assays, caspase-3/7 assays, silver staining, gelatin zymography and Western blotting were performed. Hypoxia led to morphological signs of cell damage and increased the release of LDH in cultures containing sera T0 (P < 0.01) and T1 (P < 0.05), but not sera T2, which reduced the hypoxia-mediated LDH release compared with sera T0 (P < 0.05). Gelatin zymography revealed a significant reduction of activities of the matrixmetalloproteinase (MMP)-2 and MMP-9 in the protective sera T2 compared with the nonprotective sera T0 (MMP-2: P < 0.01; MMP-9: P < 0.05). Addition of human recombinant MMP-2 and MMP-9 to MMP-deficient culture media increased the sensitivity of CaCo-2 cells to hypoxia-induced cell damage (P < 0.05), but did not result in a reduced phosphorylation of prosurvival kinases p42/44 and protein kinase B (Akt) or increased activity of caspase-3/7. Our results suggest MMP-2 and MMP-9 as currently unknown humoral factors that may be involved in RIPC-mediated cytoprotection in the intestine.
Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany.
Karina
K
Zitta
Patrick
P
Meybohm
Berthold
B
Bein
Christin
C
Heinrich
Jochen
J
Renner
Jochen
J
Cremer
Markus
M
Steinfath
Jens
J
Scholz
Martin
M
Albrecht
D015153:Blotting, Western; D018938:Caco-2 Cells; D015687:Cell Hypoxia / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007422:Intestines / Q000166:cytology*; D019194:Ischemic Preconditioning; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum / Q000378:metabolism*
D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9