Detection of Apoptosis for the Noninvasive Diagnosis of Cardiac Allograft Rejection

  • Francis G. Blankenberg
  • Jagat Narula
  • Johnathan F. Tait
  • Robert C. Robbins
  • H. William Strauss


An important feature of transplant rejection is the immune mediated death of graft myocytes and host mononuclear cells invading the graft. In this instance, a major cause of cell death is apoptosis as mentioned in previous chapters. This stereo-typed form of cell death is associated with the expression of phosphatidylserine (PS) on the surface of the apoptotic cell (most likely as a signaling mechanism to adjacent cells and phagocytes). A physiologic protein, annexin V, binds with nanomolar affinity to cell membrane bound PS, and likely plays an important role in the cell-cell signaling and local inhibition of apoptotic cell death. We describe the animal model work and the initial clinical imaging potential of radiolabeled annexin V imaging.


Annexin Versus Acute Rejection Transplant Rejection Cardiac Allograft Cardiac Allograft Rejection 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Seino K, Kayagaki N, Bashuda H, Okumura K, Yagita H. Contribution of Fas ligand to cardiac allograft rejection. Int Immuno 1996;8:1347–1354.CrossRefGoogle Scholar
  2. 2.
    Laguens RP, Cabeza Meckert PM, San Martino J, Perrone S, Favaloro R. Identification of programmed cell death (apoptosis) in situ by means of specific labeling of nuclear DNA fragments in heart biopsy samples during acute rejection episodes. J Heart Lung Transplant 1996;15:911–918.PubMedGoogle Scholar
  3. 3.
    Jollow KC, Sundstrom JB, Gravanis MB, Kanter K, Herskowitz A, Ansari AA. Apoptosis of mononuclear cell infiltrates in cardiac allograft biopsy specimens questions studies of biopsy-cultured cells. Transplantation 1997;63:1482–1489.PubMedCrossRefGoogle Scholar
  4. 4.
    Matiba B, Mariani SM, Krammer PH. The CD95 System and the death of a lymphocyte. Immunology 1997;9:59–68.Google Scholar
  5. 5.
    Bergese SD, Klenotic SM, Wakely ME, Sedmak DD, Orosz CG. Apoptosis in murine cardiac grafts. Transplantation 1997;63:320–325.PubMedCrossRefGoogle Scholar
  6. 6.
    Schultz M, Schuurman HJ, Joergensen J, et al. Acute rejection of vascular heart allografts by perforin deficient mice. Eur J Immunol 1995;25:474–480.CrossRefGoogle Scholar
  7. 7.
    Larsen CP, Alexander DZ, Henrix R, Ritchie S, Pearson TC. Fas-mediated cytotoxicity. Transplantation 1995;60:221–224.PubMedCrossRefGoogle Scholar
  8. 8.
    Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449–1456.PubMedCrossRefGoogle Scholar
  9. 9.
    Thompson BC. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–1462.PubMedCrossRefGoogle Scholar
  10. 10.
    Kageyama Y, Li X-K, Suzuki S, Suzuki H, Suzuki K, Kazui T, Harada Y. Apoptosis is involved in acute cardiac allograft rejection in rats. Annuals of Thoracic Surgery 1998;65:1604–1609.CrossRefGoogle Scholar
  11. 11.
    Szaboles MJ, Ravalli S, Mihanov O, Sciacca RR, Michler RE, Cannon PJ. Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection. Transplantation 1998;65:804–812.CrossRefGoogle Scholar
  12. 12.
    Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, Kopiwoda S, Abrams MJ, Darkes M, Robbins RC, Maecker HT, Strauss HW. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci USA, 95:6349–6354, May 1998 Medical Sciences.PubMedCrossRefGoogle Scholar
  13. 13.
    Romisch J, Paques EP. Annexins: Calcium binding proteins of multifunctional importance? Med Microbiol Immunol 1991;180:109–126.PubMedCrossRefGoogle Scholar
  14. 14.
    Rand JR. Annexinopathies—a new class of diseases. New Engl J Med 1999;340:1035–1036 (editorial).PubMedCrossRefGoogle Scholar
  15. 15.
    Bohn H. Placental DNA pregnancy proteins. In: Carcinoembryonic proteins. Lehman FG ed. Vol 1. 1979; pp 289–99. Elsevier, North Holland Biomedical Press.Google Scholar
  16. 16.
    Reutelingsperger C, Hornstra G, Hemker H. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985;151:625–629.PubMedCrossRefGoogle Scholar
  17. 17.
    Kaneko N, Matsuda R, Hosoda S, et al. Measurement of plasma annexin V by ELISA in the early detection of acute myocardial infarction. Clin Chim Acta 1996;251:65–80.PubMedCrossRefGoogle Scholar
  18. 18.
    Gidon-Jeangirard C, Solito E, Hofmann A, et al. Annexin V counteracts apoptosis while inducing Ca2+ influx in human lymphocytic T cells. Biochem Biophys Res Comm 1999;265:709–715.PubMedCrossRefGoogle Scholar
  19. 19.
    Trouvé P, Legot S, Belikova I, et al. Localization and quantification of cardiac annexins II, V, and VI in hypertensive guinea pigs. Am J Physiol 1999;276:H1159–H1166.PubMedGoogle Scholar
  20. 20.
    Tait JF, Gibson D, Fujikawa K. Phospholipid binding properties of human placental anticoagulant protein I, a member of the lipocortin family. J Biol Chem 1989;264:7944–7949.PubMedGoogle Scholar
  21. 21.
    Tait J, Gibson D. Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content. Arch Biochem Biophys 1992;298:187–191.PubMedCrossRefGoogle Scholar
  22. 22.
    Dachary-Prigent J, Freyssinet JM, Pasquet JM, Carron JC, Nurden A. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study snowing a role for free sulfhydryl groups. Blood 1993;81:2554–2565.PubMedGoogle Scholar
  23. 23.
    Tait JF, Cerqueira MD, Dewhurst TA, Fujikawa K, Ritchie JL, Stratton JR. Evaluation of annexin V as a platelet directed thrombus targeting agent. Thromb Res 1994;75:491–501.PubMedCrossRefGoogle Scholar
  24. 24.
    Stratton et al. unpublished data.Google Scholar
  25. 25.
    Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, vanOers MHJ. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415–1420.Google Scholar
  26. 26.
    Boersma AMW, Nooter K, Oostrum RG, Stoter G. Quantification of apoptotic cells with fluorescein isothiocyante labeled annexin V in Chinese hamster ovary cell cultures treated with cisplatin. Cytometry 1996;24:123–130.PubMedCrossRefGoogle Scholar
  27. 27.
    van Heerde WL, de Groot PG, Reutelingsperger CPM. The complexity of the phospholipid binding protein annexin V. Thromb and Hemostasis 1995;73:172–179.Google Scholar
  28. 28.
    Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997;89:1121–1132.PubMedGoogle Scholar
  29. 29.
    Tait JF, Smith C, Wood BL. Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood flow cytometry with annexin V. Blood Cells, Molecules, and Diseases. 1999;25:271–278.PubMedCrossRefGoogle Scholar
  30. 30.
    Naito M, Nagashima K, Mashima T, Tsuruo T. Phosphatidylserine externalization is a downstream event of interleukin-1β-converting enzyme family protease activation during apoptosis. Blood 1997;89:2060–2066.PubMedGoogle Scholar
  31. 31.
    Fadok VA, Bratton DL, Rose DM, et al. A receptor for phosphatidylserine specific clearance of apoptotic cells. Nature 2000;405:85–90.PubMedCrossRefGoogle Scholar
  32. 32.
    Allen TR, Hunter WJ, Agrawal DK. Morphological and biochemical characterization and analysis of apoptosis. J Pharm Toxic Method 1997;37:215–228.CrossRefGoogle Scholar
  33. 33.
    van England M, Nieland LJW, Ramaekers FCS, Schutte B, Reutelingsperger CPM. Annexin V-affinity assay: a review on a apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998;31:1–9.CrossRefGoogle Scholar
  34. 34.
    van den Eijnde SM, Luijsterburg AJM, Boshart L, De Zeeuw CI, van Dierendonck JH, Reutelingsperger CPM, Vermeij-Keers C. In situ detection of apoptosis during embryogenesis with annexin V: from whole mount to ultrastructure. Cytometry 1997;29:313–320.PubMedCrossRefGoogle Scholar
  35. 35.
    Blankenberg FG, Ohtsuki K, Strauss HW. Dying a thousand deaths. Radionuclide imaging of apoptosis. Quart J Nucl Med 1999;43:170–176.Google Scholar
  36. 36.
    Vriens PW, Blankenberg FG, Stoot JH, Ohtsuki K, Berry GJ, Tait HW, Strauss, Robbins RC. The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Cardiovasc Surg 1998;116:844–853.PubMedCrossRefGoogle Scholar
  37. 37.
    Kown MH, Van der Steenhoven TJ, Blankenberg FG, Hoyt G, Strauss HW, Robbins RC. Zn2+ blocks cardiac allograft apoptosis as measured by Annexin V imaging. Circulation 2000;102-Suppl: III-228-III-232.Google Scholar
  38. 38.
    D’Arceuil H, Rhine W, de Crespigny A, Yenari M, Tait JF, Strauss HW, Engelhorn T, Kastrup A, Moseley M, Blankenberg FG. 99mTc annexin V imaging of neonatal hypoxic brain injury. Stroke 2000;31:2692–2700.PubMedCrossRefGoogle Scholar
  39. 39.
    Blankenberg FG, Robbins RC, Stoot JH, Vriens PW, Berry GJ, Tait JF, Strauss HW. Radionuclide imaging of acute lung transplant rejection with annexin V. Chest 2000;117:834–840.PubMedCrossRefGoogle Scholar
  40. 40.
    Ogura Y, Krams SM, Martinez OM, Kopiwoda S, Higgins JP, Esquivel CO, Strauss HW, Tait JF, Blankenberg FG. Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation. Radiology 2000;214:795–800.PubMedGoogle Scholar
  41. 41.
    Belhocine TZ, Hustinx R, Jerusalem G, Fassotte MF, Duysinx B, Quaden C, Rigo P. 99m Tc rh-annexin V (Apomate™) as a marker of apoptosis resulting from chemotherapy: preliminary results. J Nucl Med 2000;41:263P (abstract).Google Scholar
  42. 42.
    Hofstra L, Liem IH, Dumont EA, Boersma HH, van Heerde WL, Doevendans PA, De Muinck E, Wellens HJ, Kemerink GJ, Reutelingsperger CP, Heidendal GA. Visualisation of cell death in vivo in patients with acute myocardial infarction. Lancet 2000;356:209–212.PubMedCrossRefGoogle Scholar
  43. 43.
    Strauss HW, Narula J, Blankenberg FG. Radioimaging to identify myocardial cell death and probably injury. Lancet 2000;356:180–181.PubMedCrossRefGoogle Scholar
  44. 44.
    Palojoki E, Saraste A, Eriksson A, Pulkki K, Kallajoki M, Tikkanen I, Volpio-Pulkki LM. Prolonged cardiomyocyte apoptosis in the remote myocardium is associated with left ventricular remodeling after experimental myocardial infarction. J Am Coll Cardiol 2000;35:Suppl A P39 (abstract).Google Scholar
  45. 45.
    Narula J, Acio ER, Fyfe B, Narula N, Samuels LE, Wood D, Fitzpatrick JM, Guerraty A, Tomaszewski JE, Snyder G, Kelly C, Blankenberg F, Strauss HW. Phase-I 99mTc-annexin-V imaging study in heart transplant rejection: can noninvasive detection of apoptosis in cardiac allografts rejection obviate the need for endomyocardial biopsy? Circulation 2000;102:11–769.CrossRefGoogle Scholar
  46. 46.
    Strauss HW, Kown M, Hunt S, Robbins RS, Stafford-Cecil S, Tait JF, Blankenberg FG. Blood clearance of 99mTc N2S2 annexin V in human subjects. J Nucl Med 2000;41:149P (abstract).Google Scholar
  47. 47.
    Billingham MR, Cary NRB, Hammond ME, et al. A working formulation for the standardization of nomenclaure in the diagnosis of heart and lung rejection: heart rejection study group. J Heart Transplant 1990;9:587–593.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Francis G. Blankenberg
    • 1
  • Jagat Narula
    • 1
  • Johnathan F. Tait
    • 1
  • Robert C. Robbins
    • 1
  • H. William Strauss
    • 1
  1. 1.Stanford University Medical CenterStanfordUSA

Personalised recommendations