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Transfection of the Human Heme Oxygenase Gene into Rabbit Coronary Microvessel Endothelial Cells

Protective Effect against Heme and Hemoglobin Toxicity
  • N. G. Abraham

Abstract

Heme oxygenase* (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents which may induce oxidative injury such as metals, endotoxin, heme-hemoglobin and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has anti-oxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited a ≈3.0-fold increase in enzyme activity and expressed a several-fold induction of human HO mRNA as compared to endogenous rabbit HO mRNA. Transfected and non-transfected cells expressed Factor VIII antigen and exhibited similar acetylated low density lipoprotein uptake (two important features which characterize endothelial cells) with greater than 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin (rHb) and heme as compared to non-transfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Keywords

Factor Viii Heme Oxygenase Cationic Liposome Free Heme Heme Oxygenase Activity 
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.

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References

  1. 1.
    . Shibahara, S., Muller, R. M. & Taguchi, H. (1987) J. Biol. Chem.262, 12889 – 12892.PubMedGoogle Scholar
  2. 2.
    . Mitani, K., Fujita, H., Sassa, S. & Kappas, A. (1989) Biochem. Biophys. Res. Commun.165, 437 – 441.PubMedCrossRefGoogle Scholar
  3. 3.
    . Keyse, S. M. & Tyrrell, R. M. (1991) Proc. Natl Acad. Sci. USA86, 99 – 103.CrossRefGoogle Scholar
  4. 4.
    . Taketani, S., Kohno, H., Yoshinaga, T. & Tokunaga, R. (1989) FEBS Lett. 245, 173 – 176.PubMedCrossRefGoogle Scholar
  5. 5.
    . Abraham, N. G., Lin, J. H., Schwartzman, M. L., Levere, R. D. & Shibahara, S. (1988) Int. J. Biochem.20, 543 – 558.PubMedCrossRefGoogle Scholar
  6. 6.
    . Stocker, R., Yamamoto, Y., McDonagh, A. F., Glazer, A. N. & Ames, B. N. (1987) Science235, 1043 – 1046.PubMedCrossRefGoogle Scholar
  7. 7.
    . Nath, K. A., Balla, G., Vercellotti, G. M., Balla, J., Jacob, H. S., Levitt, M. D. & Rosenberg, M. E. (1992) J. Clin. Invest.90, 267 – 270.PubMedCrossRefGoogle Scholar
  8. 8.
    . Paller, M. S. & Jacob, H. S. (1994) Proc. Natl. Acad. Sci. USA91, 7002 – 7006.PubMedCrossRefGoogle Scholar
  9. 9.
    . Stocker, R. (1990) Free Radical Research Communications9, 101 – 112.PubMedCrossRefGoogle Scholar
  10. 10.
    . McCoubrey, W. K.,Jr., Ewing, J. F. & Maines, M. D. (1992) Arch. Biochem. Biophys.295, 13 – 20.PubMedCrossRefGoogle Scholar
  11. 11.
    . Shibahara, S., Yoshizawa, M., Suzuki, H., Takeda, K., Meguro, K. & Endo, K. (1993) J. Biochem.113, 214 – 218.PubMedGoogle Scholar
  12. 12.
    . Cantoni, L., Rossi, C, Rizzardini, M., Gadina, M. & Ghezzi, P. (1991) Biochem. J.279, 891 – 894.PubMedGoogle Scholar
  13. 13.
    . Rizzardini, M., Terao, M., Falciani, F. & Cantoni, L. (1993) Biochem. J.290, 343 – 347.PubMedGoogle Scholar
  14. 14.
    . Lutton, J. D., DaSilva, J-L., Moquattash, S., Brown, A. C, Levere, R. D. & Abraham, N. G. (1992) J. Cell Biol.49, 1 – 7.Google Scholar
  15. 15.
    . Alam, J. & Zhining, D. (1992)7. J. Biol. Chem. 267, 21894–21900.PubMedGoogle Scholar
  16. 16.
    . Lavrovsky, Y., Schwartzman, M. L., Levere, R. D., Kappas, A. & Abraham, N. G. (1994) Proc. Natl. Acad. Sci. USA 91, 5987 – 5991.PubMedCrossRefGoogle Scholar
  17. 17.
    . Lim, C. S., Chapman, G. D., Gammon, R. S., Muhlestein, J. B., Bauman, R. P., Stack, R. S. & Swain, J. L. (1991) Circulation83, 2007 – 2911.PubMedGoogle Scholar
  18. 18.
    . Brigham, K. L. & Schreier, H. (1993)7. J Lipid Res. 3, 31–49.Google Scholar
  19. 19.
    . Mayhew, E. G. & Papahadjopoulos, D. (1983) in Liposomes, ed. Ostro, M. J. ( Marcel Dekker, New York ), pp. 289 – 341.Google Scholar
  20. 20.
    . Brigham, K. L., Canonico, A. E., Conary, J. T. & Meyrick, B. O. (1992) in Mediators of Sepsis, eds. Lamy, M. & Thijs, L. G. ( Springer-Verlag, New York ), pp. 393 – 405.Google Scholar
  21. 21.
    . Nabel, E. G., Plautz, G. & Nabel, G. J. (1990) Science249, 1285 – 1288.PubMedCrossRefGoogle Scholar
  22. 22.
    . Gerritsen, M. E., Carley, W. & Milici, A. J. (1988) Adv. Cell Cult.6, 35 – 67.Google Scholar
  23. 23.
    . Brenner, D. A. & Bloomer, J. R. (1980) N. Engl. J. Med.302, 765.PubMedCrossRefGoogle Scholar
  24. 24.
    . Kappas, A., Drummond, G. S., Henschke, C, Petmezaki, S. & Valaes, T. (1994) in CURRENT PROGRESS IN PERINATAL MEDICINE. The Proceedings of the 2nd World Congress of Perinatal Medicine, Rome, Italy, eds. Cosmi, E. V. & DiRenzo, G. C. ( The Parthenon Publishing Group, London ), pp. 623 – 629.Google Scholar
  25. 25.
    . Valaes, T., Petmezaki, S., Henschke, C, Drummond, G. S & Kappas, A. (1994) Pediatrics93, 1 – 11.PubMedGoogle Scholar
  26. 26.
    . Drummond, G. S., Galbraith, R. A., Sardana, M. K. & Kappas, A. (1987) Arch. Biochem. Biophys.255, 64 – 74.PubMedCrossRefGoogle Scholar
  27. 27.
    . Landaw, S. A., Drummond, G. S. & Kappas, A. (1989) Pediatrics84, 1091 – 1096.PubMedGoogle Scholar
  28. 28.
    . Drummond, G. S., Rosenberg, D. W., Kihlstrom Johanson, A. C. & Kappas, A. (1989) Pharmacology39, 273 – 284.PubMedCrossRefGoogle Scholar
  29. 29.
    . Balla, J., Jacob, H. S., Balla, G., Nath, K., Eaton, J. W. & Vercelloti, J. M. (1993) Proc. Natl. Acad. Sci. USA90, 9285 – 9290.PubMedCrossRefGoogle Scholar
  30. 30.
    . Kutty, G, Hayden, B., Osawa, Y, Wiggert, B., Chader, G. J. & Kutty, R. K. (1992) Curr. Eye Res. 11, 153 – 160.PubMedCrossRefGoogle Scholar
  31. 31.
    . Vile, G. G. & Tyrrell, R. M. (1993) J. Biol. Chem.268, 14678 – 14681.PubMedGoogle Scholar
  32. 32.
    . Neuzil, J. & Stocker, R. (1994) J. Biol. Chem. 269, 16712 – 16719.PubMedGoogle Scholar
  33. 33.
    . Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V. & Snyder, S. H. (1993) Science 259, 381 – 384.PubMedCrossRefGoogle Scholar
  34. 34.
    . Vile, G. F., Basu-Modak, S., Waltner, C. & Tyrrell, R. M. (1994) Proc. Natl. Acad. Sci. USA 91, 2607 – 2610.PubMedCrossRefGoogle Scholar
  35. 35.
    . Morishita, T, Peresleny, T, Noiri, E., Staudinger, R., Abraham, N. G. & Goligorsky, M. S. (1994) J. Am. Soc. Nephrol. 5, 904(Abstract).Google Scholar
  36. 36.
    . Erzurum, S. E., Lemarchand, P., Rosenfeld, M. A., Yoo, J-H. & Crystal, R. G. (1993) Nucleic Acids. Res. 21, 1607 – 1612.PubMedCrossRefGoogle Scholar
  37. 37.
    . Jornot, L. & Junad, A. F. (1993) Am. J. Physiol. 264, L482 – L489.PubMedGoogle Scholar
  38. 38.
    . Llesury, S., Evelson, P., Gonzales-Flecha, B., Peralta, J., Caneras, M. C, Poderoso, J. J. & Boveris, A. (1994) Free Radic. Biol. Med. 16, 445 – 451.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • N. G. Abraham
    • 1
  1. 1.The Rockefeller UniversityNew YorkUSA

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