Molecular Medicine

, Volume 18, Issue 1, pp 29–37 | Cite as

Serum from Patients Undergoing Remote Ischemic Preconditioning Protects Cultured Human Intestinal Cells from Hypoxia-Induced Damage: Involvement of Matrixmetalloproteinase-2 and -9

  • Karina Zitta
  • Patrick Meybohm
  • Berthold Bein
  • Christin Heinrich
  • Jochen Renner
  • Jochen Cremer
  • Markus Steinfath
  • Jens Scholz
  • Martin Albrecht
Research Article


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.



We thank O Broch, H Franksen, A Carstens, M Betz, C Rodde, M Jonigkeit, I Möller, D Maahs, T Schuett, F Lauer and S Schroeder for technical assistance.

Supplementary material

10020_2012_1801029_MOESM1_ESM.pdf (496 kb)
Serum from Patients Undergoing Remote Ischemic Preconditioning Protects Cultured Human Intestinal Cells from Hypoxia-Induced Damage: Involvement of Matrixmetalloproteinase-2 and -9


  1. 1.
    Kharbanda RK, Nielsen TT, Redington AN. (2009) Translation of remote ischaemic preconditioning into clinical practice. Lancet. 374:1557–65.CrossRefGoogle Scholar
  2. 2.
    Kloner RA. (2009) Clinical application of remote ischemic preconditioning. Circulation. 119:776–8.CrossRefGoogle Scholar
  3. 3.
    Tapuria N, et al. (2008) Remote ischemic preconditioning: a novel protective method from ischemia reperfusion injury: a review. J. Surg. Res. 150:304–30.CrossRefGoogle Scholar
  4. 4.
    Hausenloy DJ, Yellon DM. (2011) The therapeutic potential of ischemic conditioning: an update. Nat. Rev. Cardiol. 8:619–29.CrossRefGoogle Scholar
  5. 5.
    Sadat U. (2009) Signaling pathways of cardioprotective ischemic preconditioning. Int. J. Surg. 7:490–8.CrossRefGoogle Scholar
  6. 6.
    Korth U, et al. (2003) Intestinal ischaemia during cardiac arrest and resuscitation: comparative analysis of extracellular metabolites by micro-dialysis. Resuscitation. 58:209–17.CrossRefGoogle Scholar
  7. 7.
    Rupani B, et al. (2007) Relationship between disruption of the unstirred mucus layer and intestinal restitution in loss of gut barrier function after trauma hemorrhagic shock. Surgery. 141:481–9.CrossRefGoogle Scholar
  8. 8.
    Epstein MD, Tchervenkov JI, Alexander JW, Johnson JR, Vester JW. (1991) Increased gut permeability following burn trauma. Arch. Surg. 126:198–200.CrossRefGoogle Scholar
  9. 9.
    Roumen RM, van der Vliet JA, Wevers RA, Goris RJ. (1993) Intestinal permeability is increased after major vascular surgery. J. Vasc. Surg. 17:734–7.CrossRefGoogle Scholar
  10. 10.
    Chaudhuri N, James J, Sheikh A, Grayson AD, Fabri BM. (2006) Intestinal ischaemia following cardiac surgery: a multivariate risk model. Eur. J. Cardiothorac. Surg. 29:971–7.CrossRefGoogle Scholar
  11. 11.
    Oudemans-van Straaten HM, et al. (1996) Intestinal permeability, circulating endotoxin, and postoperative systemic responses in cardiac surgery patients. J. Cardiothorac. Vasc. Anesth. 10:187–94.CrossRefGoogle Scholar
  12. 12.
    Solligard E, et al. (2008) Gut luminal lactate measured by microdialysis mirrors permeability of the intestinal mucosa after ischemia. Shock. 29:245–51.PubMedGoogle Scholar
  13. 13.
    Sun Z, et al. (1998) The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats. Shock. 10:203–12.CrossRefGoogle Scholar
  14. 14.
    Magnotti LJ, Deitch EA. (2005) Burns, bacterial translocation, gut barrier function, and failure. J. Burn Care Rehabil. 26:383–91.CrossRefGoogle Scholar
  15. 15.
    Doig CJ, et al. (1998) Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients. Am. J. Respir. Crit. Care Med. 158:444–51.CrossRefGoogle Scholar
  16. 16.
    Swank GM, Deitch EA. (1996) Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J. Surg. 20:411–7.CrossRefGoogle Scholar
  17. 17.
    Gaussorgues P, et al. (1988) Bacteremia following cardiac arrest and cardiopulmonary resuscitation. Intensive Care Med. 14:575–7.CrossRefGoogle Scholar
  18. 18.
    Saeki I, Matsuura T, Hayashida M, Taguchi T. (2011) Ischemic preconditioning and remote ischemic preconditioning have protective effect against cold ischemia-reperfusion injury of rat small intestine. Pediatr. Surg. Int. 27:857–62.CrossRefGoogle Scholar
  19. 19.
    Liu KX, et al. (2009) Immediate postconditioning during reperfusion attenuates intestinal injury. Intensive Care Med. 35:933–42.CrossRefGoogle Scholar
  20. 20.
    Zitta K, et al. (2010) Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: findings from a human, in-vitro hypoxia model. Eur. J. Pharmacol. 645:39–46.CrossRefGoogle Scholar
  21. 21.
    Zitta K, et al. (2010) Hypoxia-induced cell damage is reduced by mild hypothermia and post-conditioning with catalase in-vitro: application of an enzyme based oxygen deficiency system. Eur. J. Pharmacol. 628:11–18.CrossRefGoogle Scholar
  22. 22.
    Meybohm P, et al. (2009) Hypothermia and post-conditioning after cardiopulmonary resuscitation reduce cardiac dysfunction by modulating inflammation, apoptosis and remodeling. PLoS One. 4:e7588.CrossRefGoogle Scholar
  23. 23.
    Butler GS, Overall CM. (2009) Updated biological roles for matrix metalloproteinases and new “intracellular” substrates revealed by degradomics. Biochemistry. 48:10830–45.CrossRefGoogle Scholar
  24. 24.
    Cauwe B, Opdenakker G. (2010) Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases. Crit. Rev. Biochem. Mol. Biol. 45:351–423.CrossRefGoogle Scholar
  25. 25.
    Hausenloy DJ, Yellon DM. (2004) New directions for protecting the heart against ischaemiareperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc. Res. 61:448–60.CrossRefGoogle Scholar
  26. 26.
    Pop C, Salvesen GS. (2009) Human caspases: activation, specificity, and regulation. J. Biol. Chem. 284:21777–81.CrossRefGoogle Scholar
  27. 27.
    Ruemmele FM, et al. (2003) Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway. Gut. 52:94–100.CrossRefGoogle Scholar
  28. 28.
    Bein B, Meybohm P. (2010) [Organ protection by conditioning]. Anasthesiol. Intensivmed. Notfallmed. Schmerzther. 45:254–61; quiz 62.CrossRefGoogle Scholar
  29. 29.
    Hausenloy DJ, Yellon DM. (2008) Remote ischaemic preconditioning: underlying mechanisms and clinical application. Cardiovasc. Res. 79:377–86.CrossRefGoogle Scholar
  30. 30.
    Woodrum DT, et al. (2009) Differential effect of 17-beta-estradiol on smooth muscle cell and aortic explant MMP2. J. Surg. Res. 155:48–53.CrossRefGoogle Scholar
  31. 31.
    Ehrlichman LK, et al. (2010) Gender-dependent differential phosphorylation in the ERK signaling pathway is associated with increased MMP2 activity in rat aortic smooth muscle cells. J. Surg. Res. 160:18–24.CrossRefGoogle Scholar
  32. 32.
    Brew K, Dinakarpandian D, Nagase H. (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim. Biophys. Acta. 1477:267–83.CrossRefGoogle Scholar
  33. 33.
    Jean-St-Michel E, et al. (2011) Remote preconditioning improves maximal performance in highly trained athletes. Med. Sci. Sports Exerc. 43:1280–6.CrossRefGoogle Scholar
  34. 34.
    Shimizu M, et al. (2009) Transient limb ischaemia remotely preconditions through a humoral mechanism acting directly on the myocardium: evidence suggesting cross-species protection. Clin. Sci. (Lond). 117:191–200.CrossRefGoogle Scholar
  35. 35.
    Page-McCaw A, Ewald AJ, Werb Z. (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat. Rev. Mol. Cell. Biol. 8:221–33.CrossRefGoogle Scholar
  36. 36.
    Zhang K, et al. (2003) HIV-induced metalloproteinase processing of the chemokine stromal cell derived factor-1 causes neurodegeneration. Nat. Neurosci. 6:1064–71.CrossRefGoogle Scholar
  37. 37.
    Choi YA, Kim DK, Bang OS, Kang SS, Jin EJ. (2009) Secretory phospholipase A2 promotes MMP-9-mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis. Biol. Cell. 102:107–19.CrossRefGoogle Scholar
  38. 38.
    Chintala SK, Zhang X, Austin JS, Fini ME. (2002) Deficiency in matrix metalloproteinase gelatinase B (MMP-9) protects against retinal ganglion cell death after optic nerve ligation. J. Biol. Chem. 277:47461–8.CrossRefGoogle Scholar
  39. 39.
    Li SJ, et al. (2010) Noninvasive limb ischemic preconditioning protects against myocardial I/R injury in rats. J. Surg. Res. 164:162–8.CrossRefGoogle Scholar
  40. 40.
    Souza DG, et al. (2007) Effects of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in a model of intestinal reperfusion injury in mice. Eur. J. Pharmacol. 571:72–80.CrossRefGoogle Scholar
  41. 41.
    Kristiansen SB, et al. (2005) Remote preconditioning reduces ischemic injury in the explanted heart by a KATP channel-dependent mechanism. Am. J. Physiol. Heart Circ. Physiol. 288:H1252–6.CrossRefGoogle Scholar
  42. 42.
    Wang L, et al. (2008) Remote ischemic preconditioning elaborates a transferable blood-borne effector that protects mitochondrial structure and function and preserves myocardial performance after neonatal cardioplegic arrest. J. Thorac. Cardiovasc. Surg. 136:335–42.CrossRefGoogle Scholar
  43. 43.
    Wolfrum S, et al. (2002) Remote preconditioning protects the heart by activating myocardial PK-Cepsilon-isoform. Cardiovasc. Res. 55:583–9.CrossRefGoogle Scholar
  44. 44.
    Hausenloy DJ, Yellon DM. (2009) Preconditioning and postconditioning: underlying mechanisms and clinical application. Atherosclerosis. 204:334–41.CrossRefGoogle Scholar

Copyright information

© The Author(s) 2012

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Karina Zitta
    • 1
  • Patrick Meybohm
    • 1
  • Berthold Bein
    • 1
  • Christin Heinrich
    • 1
  • Jochen Renner
    • 1
  • Jochen Cremer
    • 2
  • Markus Steinfath
    • 1
  • Jens Scholz
    • 1
  • Martin Albrecht
    • 1
  1. 1.Department of Anesthesiology and Intensive Care MedicineUniversity Hospital Schleswig-HolsteinKielGermany
  2. 2.Department of Heart and Vascular SurgeryUniversity Hospital Schleswig-HolsteinKielGermany

Personalised recommendations