Skip to main content

Advertisement

SpringerLink
Log in

Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences

  • Original Contribution
  • Published:
Basic Research in Cardiology Aims and scope Submit manuscript

Abstract

The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Bolli R (2001) Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33:1897–1918. doi:10.1006/jmcc.2001.1462

    Article  PubMed  CAS  Google Scholar 

  2. Bolli R (2000) The late phase of preconditioning. Circ Res 87:972–983

    PubMed  CAS  Google Scholar 

  3. Bolli R, Li QH, Tang XL, Guo Y, Xuan YT, Rokosh G, Dawn B (2007) The late phase of preconditioning and its natural clinical application—gene therapy. Heart Fail Rev 12:189–199. doi:10.1007/s10741-007-9031-4

    Article  PubMed  CAS  Google Scholar 

  4. de Belder AJ, Radomski MW, Why HJ, Richardson PJ, Bucknall CA, Salas E, Martin JF, Moncada S (1993) Nitric oxide synthase activities in human myocardium. Lancet 341:84–85 pii:0140-6736(93)92559-C

    Article  PubMed  Google Scholar 

  5. de Belder AJ, Radomski MW, Why HJ, Richardson PJ, Martin JF (1995) Myocardial calcium-independent nitric oxide synthase activity is present in dilated cardiomyopathy, myocarditis, and postpartum cardiomyopathy but not in ischaemic or valvar heart disease. Br Heart J 74:426–430

    Article  PubMed  Google Scholar 

  6. Flotte TR, Schwiebert EM, Zeitlin PL, Carter BJ, Guggino WB (2005) Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy. Hum Gene Ther 16:921–928. doi:10.1089/hum.2005.16.921

    Article  PubMed  CAS  Google Scholar 

  7. Gardin JM, Siri FM, Kitsis RN, Edwards JG, Leinwand LA (1995) Echocardiographic assessment of left ventricular mass and systolic function in mice. Circ Res 76:907–914

    PubMed  CAS  Google Scholar 

  8. Gorziglia MI, Kadan MJ, Yei S, Lim J, Lee GM, Luthra R, Trapnell BC (1996) Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol 70:4173–4178

    PubMed  CAS  Google Scholar 

  9. Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli R (1999) The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96:11507–11512

    Article  PubMed  CAS  Google Scholar 

  10. Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R (1998) Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol 275:H1375–H1387

    PubMed  CAS  Google Scholar 

  11. Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey JK, Dec GW, Semigran MJ, Rosenzweig A (1998) Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci USA 95:5251–5256

    Article  PubMed  CAS  Google Scholar 

  12. Hajjar RJ, Zsebo K (2007) AAV vectors and cardiovascular disease: targeting TNF receptor in the heart: clue to way forward with AAV? Gene Ther 14:1611–1612. doi:10.1038/sj.gt.3303047

    Article  PubMed  CAS  Google Scholar 

  13. Hausenloy DJ, Baxter G, Bell R, Botker HE, Davidson SM, Downey J, Heusch G, Kitakaze M, Lecour S, Mentzer R, Mocanu MM, Ovize M, Schulz R, Shannon R, Walker M, Walkinshaw G, Yellon DM (2010) Translating novel strategies for cardioprotection: the Hatter workshop recommendations. Basic Res Cardiol 105:677–686. doi:10.1007/s00395-010-0121-4

    Article  PubMed  Google Scholar 

  14. Haywood GA, Tsao PS, von der Leyen HE, Mann MJ, Keeling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bishopric NH, Cooke JP, McKenna WJ, Fowler MB (1996) Expression of inducible nitric oxide synthase in human heart failure. Circulation 93:1087–1094

    PubMed  CAS  Google Scholar 

  15. Heger J, Godecke A, Flogel U, Merx MW, Molojavyi A, Kuhn-Velten WN, Schrader J (2002) Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction. Circ Res 90:93–99

    Article  PubMed  CAS  Google Scholar 

  16. Henckaerts E, Linden RM (2010) Adeno-associated virus: a key to the human genome? Future Virol 5:555–574. doi:10.2217/fvl.10.48

    Article  PubMed  CAS  Google Scholar 

  17. Hester ME, Foust KD, Kaspar RW, Kaspar BK (2009) AAV as a gene transfer vector for the treatment of neurological disorders: novel treatment thoughts for ALS. Curr Gene Ther 9:428–433

    Article  PubMed  CAS  Google Scholar 

  18. Heusch G (2001) Nitroglycerin and delayed preconditioning in humans: yet another new mechanism for an old drug? Circulation 103:2876–2878

    PubMed  CAS  Google Scholar 

  19. Heusch G, Boengler K, Schulz R (2008) Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation 118:1915–1919. doi:10.1161/CIRCULATIONAHA.108.805242

    Article  PubMed  Google Scholar 

  20. Jiang H, Lillicrap D, Patarroyo-White S, Liu T, Qian X, Scallan CD, Powell S, Keller T, McMurray M, Labelle A, Nagy D, Vargas JA, Zhou S, Couto LB, Pierce GF (2006) Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs. Blood 108:107–115. doi:10.1182/blood-2005-12-5115

    Article  PubMed  CAS  Google Scholar 

  21. Jneid H, Chandra M, Alshaher M, Hornung CA, Tang XL, Leesar M, Bolli R (2005) Delayed preconditioning-mimetic actions of nitroglycerin in patients undergoing exercise tolerance tests. Circulation 111:2565–2571. doi:10.1161/CIRCULATIONAHA.104.515445

    Article  PubMed  CAS  Google Scholar 

  22. Jones JM, Wilson KH, Koch WJ, Milano CA (2002) Adenoviral gene transfer to the heart during cardiopulmonary bypass: effect of myocardial protection technique on transgene expression. Eur J Cardiothorac Surg 21:847–852 pii:S1010794002000787

    Article  PubMed  CAS  Google Scholar 

  23. Kavazis AN (2009) Exercise preconditioning of the myocardium. Sports Med 39:923–935. doi:10.2165/11317870-000000000-00000

    Article  PubMed  Google Scholar 

  24. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, Glader B, Chew AJ, Tai SJ, Herzog RW, Arruda V, Johnson F, Scallan C, Skarsgard E, Flake AW, High KA (2000) Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24:257–261. doi:10.1038/73464

    Article  PubMed  CAS  Google Scholar 

  25. Kyto V, Vuorinen T, Saukko P, Lautenschlager I, Lignitz E, Saraste A, Voipio-Pulkki LM (2005) Cytomegalovirus infection of the heart is common in patients with fatal myocarditis. Clin Infect Dis 40:683–688. doi:10.1086/427804

    Article  PubMed  Google Scholar 

  26. Lai YK, Shen WY, Brankov M, Lai CM, Constable IJ, Rakoczy PE (2002) Potential long-term inhibition of ocular neovascularization by recombinant adeno-associated virus-mediated secretion gene therapy. Gene Ther 9:804–813. doi:10.1038/sj.gt.3301695

    Article  PubMed  CAS  Google Scholar 

  27. Leesar MA, Stoddard MF, Dawn B, Jasti VG, Masden R, Bolli R (2001) Delayed preconditioning-mimetic action of nitroglycerin in patients undergoing coronary angioplasty. Circulation 103:2935–2941

    PubMed  CAS  Google Scholar 

  28. Lewis NP, Tsao PS, Rickenbacher PR, Xue C, Johns RA, Haywood GA, von der Leyen H, Trindade PT, Cooke JP, Hunt SA, Billingham ME, Valantine HA, Fowler MB (1996) Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation 93:720–729

    PubMed  CAS  Google Scholar 

  29. Li J, Wang D, Qian S, Chen Z, Zhu T, Xiao X (2003) Efficient and long-term intracardiac gene transfer in delta-sarcoglycan-deficiency hamster by adeno-associated virus-2 vectors. Gene Ther 10:1807–1813. doi:10.1038/sj.gt.33020783302078

    Article  PubMed  CAS  Google Scholar 

  30. Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, French BA (2001) Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation 103:1893–1898

    PubMed  CAS  Google Scholar 

  31. Li Q, Bolli R, Qiu Y, Tang XL, Murphree SS, French BA (1998) Gene therapy with extracellular superoxide dismutase attenuates myocardial stunning in conscious rabbits. Circulation 98:1438–1448

    PubMed  CAS  Google Scholar 

  32. Li Q, Guo Y, Ou Q, Chen N, Wu WJ, Yuan F, O’Brien E, Wang T, Luo L, Hunt GN, Zhu X, Bolli R (2011) Intracoronary administration of cardiac stem cells in mice: a new, improved technique for cell therapy in murine models. Basic Res Cardiol. doi:10.1007/s00395-011-0180-1

  33. Li Q, Guo Y, Ou Q, Cui C, Wu WJ, Tan W, Zhu X, Lanceta LB, Sanganalmath SK, Dawn B, Shinmura K, Rokosh GD, Wang S, Bolli R (2009) Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120:1222–1230. doi:10.1161/CIRCULATIONAHA.108.778688

    Article  PubMed  CAS  Google Scholar 

  34. Li Q, Guo Y, Ou Q, Wu WJ, Chen N, Zhu X, Tan W, Yuan F, Dawn B, Luo L, Hunt GN, Bolli R (2011) Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences. Basic Res Cardiol (in press)

  35. Li Q, Guo Y, Tan W, Ou Q, Wu WJ, Sturza D, Dawn B, Hunt G, Cui C, Bolli R (2007) Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-kappaB dependent pathway. Circulation 116:1577–1584. doi:10.1161/CIRCULATIONAHA.107.689810

    Article  PubMed  CAS  Google Scholar 

  36. Li Q, Guo Y, Tan W, Stein AB, Dawn B, Wu WJ, Zhu X, Lu X, Xu X, Siddiqui T, Tiwari S, Bolli R (2006) Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences. Am J Physiol Heart Circ Physiol 290:H584–H589. doi:10.1152/ajpheart.00855.2005

    Article  PubMed  CAS  Google Scholar 

  37. Li Q, Guo Y, Xuan YT, Lowenstein CJ, Stevenson SC, Prabhu SD, Wu WJ, Zhu Y, Bolli R (2003) Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism. Circ Res 92:741–748

    Article  PubMed  CAS  Google Scholar 

  38. Lim SY, Yellon DM, Hausenloy DJ (2010) The neural and humoral pathways in remote limb ischemic preconditioning. Basic Res Cardiol 105:651–655. doi:10.1007/s00395-010-0099-y

    Article  PubMed  Google Scholar 

  39. Lowenstein CJ, Glatt CS, Bredt DS, Snyder SH (1992) Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc Natl Acad Sci USA 89:6711–6715

    Article  PubMed  CAS  Google Scholar 

  40. Lowenstein PR, Castro MG (2003) Inflammation and adaptive immune responses to adenoviral vectors injected into the brain: peculiarities, mechanisms, and consequences. Gene Ther 10:946–954. doi:10.1038/sj.gt.33020483302048

    Article  PubMed  CAS  Google Scholar 

  41. Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR, Tai SJ, Ragni MV, Thompson A, Ozelo M, Couto LB, Leonard DG, Johnson FA, McClelland A, Scallan C, Skarsgard E, Flake AW, Kay MA, High KA, Glader B (2003) AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101:2963–2972. doi:10.1182/blood-2002-10-32962002-10-3296

    Article  PubMed  CAS  Google Scholar 

  42. Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ, Iwaki Y, Colosi P (1998) Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther 5:938–945. doi:10.1038/sj.gt.3300680

    Article  PubMed  CAS  Google Scholar 

  43. Melo LG, Agrawal R, Zhang L, Rezvani M, Mangi AA, Ehsan A, Griese DP, Dell’Acqua G, Mann MJ, Oyama J, Yet SF, Layne MD, Perrella MA, Dzau VJ (2002) Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation 105:602–607

    Article  PubMed  CAS  Google Scholar 

  44. Miura T, Miki T (2008) Limitation of myocardial infarct size in the clinical setting: current status and challenges in translating animal experiments into clinical therapy. Basic Res Cardiol 103:501–513. doi:10.1007/s00395-008-0743-y

    Article  PubMed  Google Scholar 

  45. Muruve DA (2004) The innate immune response to adenovirus vectors. Hum Gene Ther 15:1157–1166. doi:10.1089/hum.2004.15.1157

    Article  PubMed  CAS  Google Scholar 

  46. Okubo S, Wildner O, Shah MR, Chelliah JC, Hess ML, Kukreja RC (2001) Gene transfer of heat-shock protein 70 reduces infarct size in vivo after ischemia/reperfusion in the rabbit heart. Circulation 103:877–881

    PubMed  CAS  Google Scholar 

  47. Olivetti G, Melissari M, Capasso JM, Anversa P (1991) Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy. Circ Res 68:1560–1568

    PubMed  CAS  Google Scholar 

  48. Patel HH, Hsu A, Gross GJ (2004) Delayed cardioprotection is mediated via a non-peptide delta opioid agonist, SNC-121, independent of opioid receptor stimulation. Basic Res Cardiol 99:38–45. doi:10.1007/s00395-003-0438-3

    Article  PubMed  CAS  Google Scholar 

  49. Patel MB, Sonnenblick EH (1998) Age associated alterations in structure and function of the cardiovascular system. Am J Geriatr Cardiol 7:15–22

    PubMed  Google Scholar 

  50. Paulus WJ, Kastner S, Pujadas P, Shah AM, Drexler H, Vanderheyden M (1997) Left ventricular contractile effects of inducible nitric oxide synthase in the human allograft. Circulation 96:3436–3442

    PubMed  CAS  Google Scholar 

  51. Post H, Heusch G (2002) Ischemic preconditioning. Experimental facts and clinical perspective. Minerva Cardioangiol 50:569–605

    PubMed  CAS  Google Scholar 

  52. Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA (1991) Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 10:3941–3950

    PubMed  CAS  Google Scholar 

  53. Schulz R, Gres P, Konietzka I, Heusch G (2005) Regional differences of myocardial infarct development and ischemic preconditioning. Basic Res Cardiol 100:48–56. doi:10.1007/s00395-004-0497-5

    Article  PubMed  Google Scholar 

  54. Seppen J, Bakker C, de Jong B, Kunne C, van den Oever K, Vandenberghe K, de Waart R, Twisk J, Bosma P (2006) Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats. Mol Ther 13:1085–1092. doi:10.1016/j.ymthe.2006.01.014

    Article  PubMed  CAS  Google Scholar 

  55. Sethe S, Scutt A, Stolzing A (2006) Aging of mesenchymal stem cells. Ageing Res Rev 5:91–116. doi:10.1016/j.arr.2005.10.001

    Article  PubMed  CAS  Google Scholar 

  56. Szelid Z, Pokreisz P, Liu X, Vermeersch P, Marsboom G, Gillijns H, Pellens M, Verbeken E, Van de Werf F, Collen D, Janssens SP (2010) Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial reperfusion injury. Basic Res Cardiol 105:169–179. doi:10.1007/s00395-009-0077-4

    Article  PubMed  CAS  Google Scholar 

  57. Thielmann M, Kottenberg E, Boengler K, Raffelsieper C, Neuhaeuser M, Peters J, Jakob H, Heusch G (2010) Remote ischemic preconditioning reduces myocardial injury after coronary artery bypass surgery with crystalloid cardioplegic arrest. Basic Res Cardiol 105:657–664. doi:10.1007/s00395-010-0104-5

    Article  PubMed  CAS  Google Scholar 

  58. Umar S, van der Laarse A (2010) Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart. Mol Cell Biochem 333:191–201. doi:10.1007/s11010-009-0219-x

    Article  PubMed  CAS  Google Scholar 

  59. Vejlstrup NG, Bouloumie A, Boesgaard S, Andersen CB, Nielsen-Kudsk JE, Mortensen SA, Kent JD, Harrison DG, Busse R, Aldershvile J (1998) Inducible nitric oxide synthase (iNOS) in the human heart: expression and localization in congestive heart failure. J Mol Cell Cardiol 30:1215–1223. doi:10.1006/jmcc.1998.0686

    Article  PubMed  CAS  Google Scholar 

  60. Wright JF, Qu G, Tang C, Sommer JM (2003) Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector. Curr Opin Drug Discov Devel 6:174–178

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported in part by NIH grants R01 HL55757, HL-70897, HL-76794, and P01HL78825.

Author information

Authors and Affiliations

  1. Division of Cardiovascular Medicine, Institute of Molecular Cardiology, University of Louisville, 550 S Jackson Street, Louisville, KY, 40292, USA

    Qianhong Li, Yiru Guo, Wen-Jian Wu, Qinghui Ou, Xiaoping Zhu, Wei Tan, Fangping Yuan, Ning Chen, Buddhadeb Dawn, Li Luo, Erin O’Brien & Roberto Bolli

  2. Cardiovascular Research Institute, University of Kansas Medical Center, Kansas City, KS, 66160, USA

    Buddhadeb Dawn

Authors
  1. Qianhong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yiru Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wen-Jian Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qinghui Ou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoping Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wei Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fangping Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ning Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Buddhadeb Dawn
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Li Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Erin O’Brien
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Roberto Bolli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roberto Bolli.

Additional information

Q. Li, Y. Guo contributed equally to this study.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Q., Guo, Y., Wu, WJ. et al. Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences. Basic Res Cardiol 106, 1355–1366 (2011). https://doi.org/10.1007/s00395-011-0207-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00395-011-0207-7

Keywords

Search

Navigation