Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1998 May 1;101(9):1821–1826. doi: 10.1172/JCI1830

Protection from reoxygenation injury by inhibition of rac1.

K S Kim 1, K Takeda 1, R Sethi 1, J B Pracyk 1, K Tanaka 1, Y F Zhou 1, Z X Yu 1, V J Ferrans 1, J T Bruder 1, I Kovesdi 1, K Irani 1, P Goldschmidt-Clermont 1, T Finkel 1
PMCID: PMC508766  PMID: 9576744

Abstract

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

Full Text

The Full Text of this article is available as a PDF (451.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  2. Bogoyevitch M. A., Gillespie-Brown J., Ketterman A. J., Fuller S. J., Ben-Levy R., Ashworth A., Marshall C. J., Sugden P. H. Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion. Circ Res. 1996 Aug;79(2):162–173. doi: 10.1161/01.res.79.2.162. [DOI] [PubMed] [Google Scholar]
  3. Bolli R., Jeroudi M. O., Patel B. S., DuBose C. M., Lai E. K., Roberts R., McCay P. B. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4695–4699. doi: 10.1073/pnas.86.12.4695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolli R., Patel B. S., Jeroudi M. O., Lai E. K., McCay P. B. Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone. J Clin Invest. 1988 Aug;82(2):476–485. doi: 10.1172/JCI113621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cathcart R., Schwiers E., Ames B. N. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem. 1983 Oct 1;134(1):111–116. doi: 10.1016/0003-2697(83)90270-1. [DOI] [PubMed] [Google Scholar]
  6. Chinnadurai G., Chinnadurai S., Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol. 1979 Nov;32(2):623–628. doi: 10.1128/jvi.32.2.623-628.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Downey J. M. Free radicals and their involvement during long-term myocardial ischemia and reperfusion. Annu Rev Physiol. 1990;52:487–504. doi: 10.1146/annurev.ph.52.030190.002415. [DOI] [PubMed] [Google Scholar]
  8. Granger D. N., Korthuis R. J. Physiologic mechanisms of postischemic tissue injury. Annu Rev Physiol. 1995;57:311–332. doi: 10.1146/annurev.ph.57.030195.001523. [DOI] [PubMed] [Google Scholar]
  9. Griendling K. K., Minieri C. A., Ollerenshaw J. D., Alexander R. W. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994 Jun;74(6):1141–1148. doi: 10.1161/01.res.74.6.1141. [DOI] [PubMed] [Google Scholar]
  10. Gulbins E., Brenner B., Schlottmann K., Welsch J., Heinle H., Koppenhoefer U., Linderkamp O., Coggeshall K. M., Lang F. Fas-induced programmed cell death is mediated by a Ras-regulated O2- synthesis. Immunology. 1996 Oct;89(2):205–212. doi: 10.1046/j.1365-2567.1996.d01-743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guzman R. J., Hirschowitz E. A., Brody S. L., Crystal R. G., Epstein S. E., Finkel T. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10732–10736. doi: 10.1073/pnas.91.22.10732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guzman R. J., Lemarchand P., Crystal R. G., Epstein S. E., Finkel T. Efficient and selective adenovirus-mediated gene transfer into vascular neointima. Circulation. 1993 Dec;88(6):2838–2848. doi: 10.1161/01.cir.88.6.2838. [DOI] [PubMed] [Google Scholar]
  13. Irani K., Xia Y., Zweier J. L., Sollott S. J., Der C. J., Fearon E. R., Sundaresan M., Finkel T., Goldschmidt-Clermont P. J. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science. 1997 Mar 14;275(5306):1649–1652. doi: 10.1126/science.275.5306.1649. [DOI] [PubMed] [Google Scholar]
  14. Johnson N. L., Gardner A. M., Diener K. M., Lange-Carter C. A., Gleavy J., Jarpe M. B., Minden A., Karin M., Zon L. I., Johnson G. L. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. J Biol Chem. 1996 Feb 9;271(6):3229–3237. doi: 10.1074/jbc.271.6.3229. [DOI] [PubMed] [Google Scholar]
  15. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  16. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  17. Knight R. J., Buxton D. B. Stimulation of c-Jun kinase and mitogen-activated protein kinase by ischemia and reperfusion in the perfused rat heart. Biochem Biophys Res Commun. 1996 Jan 5;218(1):83–88. doi: 10.1006/bbrc.1996.0016. [DOI] [PubMed] [Google Scholar]
  18. Koong A. C., Chen E. Y., Mivechi N. F., Denko N. C., Stambrook P., Giaccia A. J. Hypoxic activation of nuclear factor-kappa B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2). Cancer Res. 1994 Oct 15;54(20):5273–5279. [PubMed] [Google Scholar]
  19. Laderoute K. R., Webster K. A. Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes. Circ Res. 1997 Mar;80(3):336–344. doi: 10.1161/01.res.80.3.336. [DOI] [PubMed] [Google Scholar]
  20. Lee S. W., Trapnell B. C., Rade J. J., Virmani R., Dichek D. A. In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries. Circ Res. 1993 Nov;73(5):797–807. doi: 10.1161/01.res.73.5.797. [DOI] [PubMed] [Google Scholar]
  21. Lindsay T., Walker P. M., Mickle D. A., Romaschin A. D. Measurement of hydroxy-conjugated dienes after ischemia-reperfusion in canine skeletal muscle. Am J Physiol. 1988 Mar;254(3 Pt 2):H578–H583. doi: 10.1152/ajpheart.1988.254.3.H578. [DOI] [PubMed] [Google Scholar]
  22. Lo Y. Y., Cruz T. F. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem. 1995 May 19;270(20):11727–11730. doi: 10.1074/jbc.270.20.11727. [DOI] [PubMed] [Google Scholar]
  23. Lo Y. Y., Wong J. M., Cruz T. F. Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases. J Biol Chem. 1996 Jun 28;271(26):15703–15707. doi: 10.1074/jbc.271.26.15703. [DOI] [PubMed] [Google Scholar]
  24. McGrory W. J., Bautista D. S., Graham F. L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. doi: 10.1016/0042-6822(88)90302-9. [DOI] [PubMed] [Google Scholar]
  25. Meier B., Radeke H. H., Selle S., Younes M., Sies H., Resch K., Habermehl G. G. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem J. 1989 Oct 15;263(2):539–545. doi: 10.1042/bj2630539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moore K. A., Sethi R., Doanes A. M., Johnson T. M., Pracyk J. B., Kirby M., Irani K., Goldschmidt-Clermont P. J., Finkel T. Rac1 is required for cell proliferation and G2/M progression. Biochem J. 1997 Aug 15;326(Pt 1):17–20. doi: 10.1042/bj3260017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morooka H., Bonventre J. V., Pombo C. M., Kyriakis J. M., Force T. Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from the c-jun promoter. J Biol Chem. 1995 Dec 15;270(50):30084–30092. doi: 10.1074/jbc.270.50.30084. [DOI] [PubMed] [Google Scholar]
  28. Mukhopadhyay D., Tsiokas L., Zhou X. M., Foster D., Brugge J. S., Sukhatme V. P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 1995 Jun 15;375(6532):577–581. doi: 10.1038/375577a0. [DOI] [PubMed] [Google Scholar]
  29. Ohba M., Shibanuma M., Kuroki T., Nose K. Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol. 1994 Aug;126(4):1079–1088. doi: 10.1083/jcb.126.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olson M. F., Ashworth A., Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995 Sep 1;269(5228):1270–1272. doi: 10.1126/science.7652575. [DOI] [PubMed] [Google Scholar]
  31. Pombo C. M., Bonventre J. V., Avruch J., Woodgett J. R., Kyriakis J. M., Force T. The stress-activated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion. J Biol Chem. 1994 Oct 21;269(42):26546–26551. [PubMed] [Google Scholar]
  32. Rao K. M., Padmanabhan J., Kilby D. L., Cohen H. J., Currie M. S., Weinberg J. B. Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor. J Leukoc Biol. 1992 May;51(5):496–500. doi: 10.1002/jlb.51.5.496. [DOI] [PubMed] [Google Scholar]
  33. Seko Y., Tobe K., Takahashi N., Kaburagi Y., Kadowaki T., Yazaki Y. Hypoxia and hypoxia/reoxygenation activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes. Biochem Biophys Res Commun. 1996 Sep 13;226(2):530–535. doi: 10.1006/bbrc.1996.1389. [DOI] [PubMed] [Google Scholar]
  34. Shibanuma M., Kuroki T., Nose K. Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells. Oncogene. 1990 Jul;5(7):1025–1032. [PubMed] [Google Scholar]
  35. Sulciner D. J., Irani K., Yu Z. X., Ferrans V. J., Goldschmidt-Clermont P., Finkel T. rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation. Mol Cell Biol. 1996 Dec;16(12):7115–7121. doi: 10.1128/mcb.16.12.7115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sundaresan M., Yu Z. X., Ferrans V. J., Irani K., Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995 Oct 13;270(5234):296–299. doi: 10.1126/science.270.5234.296. [DOI] [PubMed] [Google Scholar]
  37. Sundaresan M., Yu Z. X., Ferrans V. J., Sulciner D. J., Gutkind J. S., Irani K., Goldschmidt-Clermont P. J., Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J. 1996 Sep 1;318(Pt 2):379–382. doi: 10.1042/bj3180379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verheij M., Bose R., Lin X. H., Yao B., Jarvis W. D., Grant S., Birrer M. J., Szabo E., Zon L. I., Kyriakis J. M. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 1996 Mar 7;380(6569):75–79. doi: 10.1038/380075a0. [DOI] [PubMed] [Google Scholar]
  39. Xia Z., Dickens M., Raingeaud J., Davis R. J., Greenberg M. E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995 Nov 24;270(5240):1326–1331. doi: 10.1126/science.270.5240.1326. [DOI] [PubMed] [Google Scholar]
  40. Zweier J. L., Flaherty J. T., Weisfeldt M. L. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1404–1407. doi: 10.1073/pnas.84.5.1404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zweier J. L., Kuppusamy P., Thompson-Gorman S., Klunk D., Lutty G. A. Measurement and characterization of free radical generation in reoxygenated human endothelial cells. Am J Physiol. 1994 Mar;266(3 Pt 1):C700–C708. doi: 10.1152/ajpcell.1994.266.3.C700. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES