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Vascular Gene Transfer of the Human Inducible Nitric Oxide Synthase: Characterization of Activity and Effects on Myointimal Hyperplasia

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Abstract

Background

Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible NO synthase (iNOS) gene transfer even at low efficiency will provide adequate local NO production to achieve this goal.

Materials and Methods

A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries?

Results

DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 ± 10.7 nmol/mg protein/24 h vs. 5.5 ± 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 ± 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 ± 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4 dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect.

Conclusions

These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4 may be required.

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References

  1. Ross R. (1993) The pathogenesis of atherosclerosis: A perspective for the 1990’s. Nature 362: 801–809.

    Article  CAS  PubMed  Google Scholar 

  2. Powel JS, Clozel JP, Muller RK, et al. (1989) Inhibitors of angiotension-converting enzyme prevent myointimal proliferation after vascular injury. Science 245: 186–188.

    Article  Google Scholar 

  3. Simons M, Edelman ER, DeKeyser JL, Langer RS, Rosenberg RD. (1992) Antisense c-myb oligonucleotides suppress smooth muscle accumulation in vivo. Nature 359: 67–70.

    Article  CAS  PubMed  Google Scholar 

  4. Ohno T, Gordon D, San H, et al. (1994) Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science 265: 781–784.

    Article  CAS  PubMed  Google Scholar 

  5. Chang MW, Barr E, Seltzer J, et al. (1995) Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 267: 518–522.

    Article  CAS  PubMed  Google Scholar 

  6. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl Acad. Sci. U.S.A. 84: 9265–9269.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Palmer RMJ, Ferrige AG, Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524–527.

    Article  CAS  PubMed  Google Scholar 

  8. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecarvo EG, Hyman AL, Kadowitz PJ. (1981) Evidence for the inhibitory role of guanosine 3′:5′-monophosphate in ADP induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 57: 946–955.

    PubMed  CAS  Google Scholar 

  9. Garg UC, Hassid A. (1989) Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J. Clin. Invest. 83: 1774–1777.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kubes P, Suzuki M, Granger DN. (1991) Nitric oxide: An endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. U.S.A. 88: 4651–4655.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mayer B, Schmidt K, Humbert P, Böhme E. (1989) Biosynthesis of endothelium-derived relaxing factor: A cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependency converts L-arginine into an activation of soluble guanylyl cyclase. Biochem. Biophys. Res. Commun. 164: 678–685.

    Article  CAS  PubMed  Google Scholar 

  12. Busse R, Mülsch A. (1990) Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 265: 133–136.

    Article  CAS  PubMed  Google Scholar 

  13. Linder L, Kiowski W, Buhler FR, Luscher TF. (1990) Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo. Blunted response in essential hypertension. Circulation 81: 1762–1767.

    Article  CAS  PubMed  Google Scholar 

  14. Panza JA, Quyyumi AA, Brush Jr JE, Epstein SE. (1990) Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N. Engl. J. Med. 323: 22–27.

    Article  CAS  PubMed  Google Scholar 

  15. O’Donohoe MK, Murchan PM, Marks P, Feely J, Feeley TM. (1993) Endothelium-derived relaxing factor is absent in experimental in situ vein grafts. Eur. J. Vasc. Surg. 7: 144–150.

    Article  PubMed  Google Scholar 

  16. Chester AH, O’Neil GS, Moncada S, Tadjkarimi S, Yacoub MH. (1990) Low basal and stimulated release of nitric oxide in atherosclerotic epicardial coronary arteries. Lancet 336: 897–900.

    Article  CAS  PubMed  Google Scholar 

  17. McNamara DB, Bedi B, Aurora H, et al. (1993) L-arginine inhibits balloon catheter-induced intimal hyperplasia. Biochem. Biophys. Res. Commun. 193: 291–296.

    Article  CAS  PubMed  Google Scholar 

  18. Davies MG, Kim JH, Dalen H, Makhoul RG, Svendsen E, Hagen PO. (1994) Reduction of experimental vein graft intimal hyperplasia and preservation of nitric oxide-mediated relaxation by the nitric oxide precursor L-arginine. Surgery 116: 557–568.

    PubMed  CAS  Google Scholar 

  19. von der Leyen HE, Gibbons GH, Morishita R, et al. (1995) Gene therapy inhibiting neointimal vascular lesions: In vivo transfer of endothelial cell nitric oxide synthase gene. Proc. Natl. Acad. Sci. U.S.A. 92: 1137–1141.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Morris Jr SM, Billiar TR. (1994) New insights into the regulation of inducible nitric oxide synthesis. Am. J. Physiol. 266: E829–E839.

    Article  CAS  PubMed  Google Scholar 

  21. Werner-Felmayer E, Werner ER, Fuchs D, Hausen A, Reibnegger G, Wachter H. (1990) Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J. Exp. Med. 172: 1599–1607.

    Article  CAS  PubMed  Google Scholar 

  22. Geller DA, Lowenstein CJ, Shapiro RA, et al. (1993) Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90: 3491–3495.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dranoff G, Jaffee EM, Lazenby A, et al. (1993) Vaccination with irradiated tumor cells engineered to secrete murine GMCSF stimulates potent, specific and long lasting antitumor immunity. Proc. Natl. Acad. Sci. U.S.A. 90: 3539–3543.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zitvogel L, Tahara H, Cai Q, et al. (1994) Construction and characterization of retroviral vectors expressing biologically-active human interleukin-12. Human Gene Ther. 5: 1493–1506.

    Article  CAS  Google Scholar 

  25. Price J, Turner D, Cepko C. (1987) Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84: 156–160.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pear WS, Nolan GP, Scott ML, Baltimore D. (1993) Production of high titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. U.S.A. 90: 8392–8396.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Danos O, Mulligan RC. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85: 6460–6464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Danos O. (1991) Construction of retroviral packaging cell lines. In: Collins M (ed). Methods in Molecular Biology, Vol. 5: Practical Molecular Virology, Viral Vectors for Gene Expression. Humana Press, Clifton, NJ, pp. 17–27.

    Google Scholar 

  29. Davies P, Patton W. (1994) Peripheral and central vascular smooth muscle cells from rat lung exhibit different cytoskeletal protein profiles but similar growth factor requirements. J. Cell. Physiol. 159: 399–406.

    Article  CAS  PubMed  Google Scholar 

  30. Chomczynski P, Sacchi N. (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.

    Article  CAS  PubMed  Google Scholar 

  31. Geller DA, Nussler AK, DiSilvio M, et al. (1993) Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90: 522–526.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Luss H, DiSilvio M, Litton AL, Molina y Vedia L, Nussler AK, Billiar TR. (1994) Inhibition of nitric oxide synthesis enhances the expression of inducible nitric oxide synthase mRNA and protein in a model of chronic liver inflammation. Biochem. Biophys. Res. Commun. 204-2: 635–640.

    Article  Google Scholar 

  33. Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

  34. Billiar TR, Curran RD, Stuehr DJ, West MA, Bentz BG, Simmons RL. (1989) An L-argi-nine-dependent mechanism mediates Kupffer cell inhibition of hepatocyte protein synthesis in vitro. J. Exp. Med. 169: 1467–1472.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Takeshita S, Gal D, Leclerc G, et al. (1994) Increased gene expression after liposomemediated arterial gene transfer associated with intimal smooth muscle cell proliferation: In vitro and in vivo findings in a rabbit model of vascular injury. J. Clin. Invest. 93: 652–661.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Baek KJ, Thiel BA, Lucas S, Stuehr DJ. (1993) Macrophage nitric oxide synthase subunits: Purification, characterization, and role of prosthetic groups and substrates in modulating their association into a dimeric enzyme. J. Biol. Chem. 268: 21120–21129.

    PubMed  CAS  Google Scholar 

  37. Tzeng E, Billiar TR, Robbins PD, Loftus M, Stuehr DJ. (1995) Expression of human inducible NO synthase in a tetrahydrobiopterin-deficient cell line: tetrahydrobiopterin promotes assembly of enzyme subunits into an active dimer. Proc. Natl. Acad. Sci. U.S.A. 92: 11771–11775.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Croen KD. (1993) Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex type 1 replication. J. Clin. Invest. 91: 2446–2456.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hansson GK, Geng Y, Holm J, Hardhammar P, Wennmalm A, Jennische E. (1994) Arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury. J. Exp. Med. 180: 733–738.

    Article  CAS  PubMed  Google Scholar 

  40. Kaufman S. (1963) The structure of phenylalanine hydroxylation cofactor. Proc. Natl. Acad. Sci. U.S.A. 50: 1085–1093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nakayama DK, Geller DA, DiSilvio M, et al. (1994) Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle. Am. J. Physiol. 266: L455–L460.

    Article  CAS  PubMed  Google Scholar 

  42. Lancaster Jr JR. (1994) Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 91: 8137–8141.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Strauss BH, Chisholm RJ, Keeley FW, Gotleib Al, Logan RA, Armstrong PW. (1994) Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ. Res. 75: 650–658.

    Article  CAS  PubMed  Google Scholar 

  44. Curran RD, Ferrari FK, Kispert PH, et al. (1991) Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. FASEB J. 5: 2085–2092.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We would like to acknowledge the excellent technical assistance of Ms. Lori McKenzie, Ms. Solthi Tran, and Mr. Paul Moyer, as well as Dr. Laurence Zitvogel for helpful discussion. This work was supported in part by National Institutes of Health Grants GM-44100 (TRB), GM-37753 (RLS), DK-44935 (PDR), HL-32154 (BRP), and F32-GM-16645 (ET). TRB is the recipient of the George H. A. Clowes Jr., M.D., Memorial Research Career Development Award of the American College of Surgeons.

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Authors and Affiliations

  1. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Edith Tzeng, Larry L. Shears II, David A. Geller, Richard L. Simmons & Timothy R. Billiar

  2. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Paul D. Robbins & Richard L. Simmons

  3. Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Bruce R. Pitt

  4. Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Simon C. Watkins

  5. University of Pittsburgh School of Medicine, A1010, Presbyterian Hospital, Pittsburgh, Pennsylvania, 15213, USA

    Timothy R. Billiar

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Tzeng, E., Shears, L.L., Robbins, P.D. et al. Vascular Gene Transfer of the Human Inducible Nitric Oxide Synthase: Characterization of Activity and Effects on Myointimal Hyperplasia. Mol Med 2, 211–225 (1996). https://doi.org/10.1007/BF03401618

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