Transposon-mediated Gene Therapy improves Pulmonary Hemodynamics and attenuates Right Ventricular Hypertrophy: eNOS gene therapy reduces Pulmonary vascular remodeling and Arterial wall hyperplasia
Reporter: Aviva Lev-Ari, PhD, RN
Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats
*Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida, USA;
†Division of Pulmonary Medicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; and
‡Medical Research Service, Department of Veteran Affairs Medical Center, Gainesville, Florida, USA
- ↵Correspondence: 1Correspondence: Department of Pharmacology and Therapeutics, 1600 S.W. Archer Rd., Box 100267, University of Florida, College of Medicine, Gainesville, FL 32610-0267, USA. E-mail: bsf@pharmacology.ufl.edu
DISCUSSION
Despite the diverse origins of etiology of pulmonary hypertension, the various disorders share similar histological and pathological findings, including endothelial dysfunction and the proliferation of SMCs resulting in vascular remodeling, in situ thrombus formation with obliteration of distal arterioles, and an inflammatory type reaction (2)⇓ . Treatment strategies for PH have relied on the use of vasodilators (e.g., calcium-channel blockers, prostacyclin) or phosphodiesterase inhibitors (e.g., sildenafil), which promote smooth muscle relaxation (4)⇓ . While pharmacological agents can be effective, potential drawbacks include the need for continuous i.v. infusion of prostacyclin derivatives or the use of nonselective vasodilators with potential side effects (3)⇓ . Inhaled NO has also been used as a treatment in patients with PH (41)⇓ ; however, its shortcomings include minimal response rates (∼10%), expense, the need for sophisticated delivery systems, and rebound hypertension (42)⇓ . These obstacles limit the therapeutic potential of the pharmacological approaches and suggest that alternative treatment modalities should be investigated.
As an alternative to simply promoting vasodilatation, an ideal strategy would be to combat the pathological processes that drive the increased pulmonary vascular resistance and loss of pulmonary microvasculature. This includes SMC proliferation and vascular remodeling, oxidative stress, inflammatory responses, and abnormal levels of vasoconstrictive molecules such as endothelin-1 (ET-1) (43)⇓ and certain prostanoids (44⇓ , 45)⇓ .
Gene therapy, especially multigene delivery, offers the possibility to overcome some of these pathological factors by using proteins or other genetic elements, such as RNA interference (RNAi), which target key regulators of vascular tone and regeneration. A growing body of literature points to the importance of endothelial-derived NO in promoting endothelial health and regulating vascular tone and regeneration.
Therefore, overexpression of eNOS, potentially in combination with inhibitors of expression of vasoconstrictor molecules (such as ET-1), is a therapeutic strategy that may reverse some of the pathological changes associated with late-stage PH.
In the present study, a severe model of PH (monocrotaline-induced) was used to test the ability of a nonviral approach to alleviate the pathological events leading to PH. Intravenous gene delivery of plasmid DNA complexed to the synthetic polymer polyethylenimine tends to transfect endothelial cells and type II pneumocytes within the lung (31⇓ , 32⇓ , 46)⇓ . Although endothelial cells would be ideal targets, we chose to use a very active nonspecific promoter to obtain the highest level of eNOS expression possible within the lung tissue. Using the CMV-driven eNOS transposon, we could demonstrate increased eNOS protein and nitrate production in vivo following gene transfer. In theory, increased NO production should lead to SMC relaxation, vasodilatation, and a reduction in PABP, which was observed in the hemodynamic studies (Fig. 3)⇓ .
However, a key factor in PH progression is increased pulmonary resistance due to SMC proliferation, intimal wall hyperplasia, and increased wall thickness. The histological data suggest that transposon-based eNOS expression prevented this hyperplasia and vascular remodeling. As NO has the ability to both inhibit SMCs proliferation and induce apoptosis (15⇓ , 47⇓ , 48)⇓ , it was unclear if the improvement in vascular remodeling was the result of growth inhibition or apoptotic effects of NO on SMCs. Tunnel assays on the histological sections revealed no significant difference in the amount of apoptosis in gene therapy-treated animals (data not shown), suggesting the effect was more on inhibition of SMC proliferation. Taken together, these results suggest that the
transposon-based approach can increase pulmonary NO production, reduce PABP, and attenuate right ventricular hypertrophy by preventing SMC proliferation and vascular remodeling.
Although SB has been used in other animal paradigms, this is the first report of using SB-mediated gene delivery to treat PH. Benefits of this approach, compared with several previous studies using adenovirus, include its nonviral delivery method, lack of inflammatory responses to viral components, cost-effectiveness, and ability to promote sustained therapeutic transgene expression. Given that SB transposons integrate within the host genome, there is some concern this approach may induce tumorigenic mutations, as has been seen with retrovirus (49)⇓ . Although this concern may be valid, SB is still considered one of the safest integrating vectors because of its near-random nature of integration (50)⇓ .
The problems associated with SB-mediated insertional mutagenesis could be overcome through the development of transposases with site-specific integration (51)⇓ . Lastly, clinically relevant delivery methods of plasmid DNA are still needed. Although the polymer PEI has recently been used in humans (52)⇓ , the efficiency of nonviral gene transfer could be improved through the synthesis more effective liposomes (e.g., cationic polymers and lipid) or lipoplexes with reduced toxicity. These complexes must be stable within plasma, transfect the pulmonary vasculature efficiently, and be able to navigate the cytoplasm to deliver the plasmid cargo to the nucleus. Given that few long-term treatment options,
other than lung transplantation, are available for PH, the success of this nonviral gene-based approach to attenuate the pathological processes driving PH warrants further investigations.
REFERENCES
- ↵
Machado, R. D., Pauciulo, M. W., Thomson, J. R., Lane, K. B., Morgan, N. V., Wheeler, L., Phillips, J. A., 3rd, Newman, J., Williams, D., Galie, N., et al (2001) BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension. Am. J. Hum. Genet. 68,92-102Epub 2000 Dec 2012
- ↵
Hampl, V., Herget, J. (2000) Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension. Physiol. Rev. 80,1337-1372
- ↵
- ↵
Strange, J. W., Wharton, J., Phillips, P. G., Wilkins, M. R. (2002) Recent insights into the pathogenesis and therapeutics of pulmonary hypertension. Clin. Sci. (Lond). 102,253-268
- ↵
- ↵
Tyler, R. C., Muramatsu, M., Abman, S. H., Stelzner, T. J., Rodman, D. M., Bloch, K. D., McMurtry, I. F. (1999) Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am. J. Physiol. 276,L297-L303
- ↵
Champion, H. C., Bivalacqua, T. J., Greenberg, S. S., Giles, T. D., Hyman, A. L., Kadowitz, P. J. (2002) Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 99,13248-13253Epub 12002 Sep 13217
- ↵
Kouyoumdjian, C., Adnot, S., Levame, M., Eddahibi, S., Bousbaa, H., Raffestin, B. (1994) Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J. Clin. Invest. 94,578-584
- ↵
Roberts, J. D., Jr, Chiche, J. D., Weimann, J., Steudel, W., Zapol, W. M., Bloch, K. D. (2000) Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups. Circ. Res. 87,140-145
- ↵
Champion, H. C., Bivalacqua, T. J., D’Souza, F. M., Ortiz, L. A., Jeter, J. R., Toyoda, K., Heistad, D. D., Hyman, A. L., Kadowitz, P. J. (1999) Gene transfer of endothelial nitric oxide synthase to the lung of the mouse in vivo. Effect on agonist-induced and flow-mediated vascular responses. Circ. Res. 84,1422-1432
- ↵
Budts, W., Pokreisz, P., Nong, Z., Van Pelt, N., Gillijns, H., Gerard, R., Lyons, R., Collen, D., Bloch, K. D., Janssens, S. (2000) Aerosol gene transfer with inducible nitric oxide synthase reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 102,2880-2885
- ↵
Champion, H. C., Bivalacqua, T. J., Toyoda, K., Heistad, D. D., Hyman, A. L., Kadowitz, P. J. (2000) In vivo gene transfer of prepro-calcitonin gene-related peptide to the lung attenuates chronic hypoxia-induced pulmonary hypertension in the mouse. Circulation 101,923-930
- ↵
- ↵
Partovian, C., Adnot, S., Raffestin, B., Louzier, V., Levame, M., Mavier, I. M., Lemarchand, P., Eddahibi, S. (2000) Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats. Am. J. Respir. Cell Mol. Biol. 23,762-771
- ↵
- ↵
Von der Leyen, H. E., Dzau, V. J. (2001) Therapeutic potential of nitric oxide synthase gene manipulation. Circulation 103,2760-2765
- ↵
- ↵
- ↵
- ↵
Tahara, N., Kai, H., Niiyama, H., Mori, T., Sugi, Y., Takayama, N., Yasukawa, H., Numaguchi, Y., Matsui, H., Okumura, K., Imaizumi, T. (2004) Repeated gene transfer of naked prostacyclin synthase plasmid into skeletal muscles attenuates monocrotaline-induced pulmonary hypertension and prolongs survival in rats. Hum. Gene Ther. 15,1270-1278
- ↵
Campbell, A. I., Zhao, Y., Sandhu, R., Stewart, D. J. (2001) Cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension. Circulation 104,2242-2248
- ↵
Zhao, Y. D., Campbell, A. I., Robb, M., Ng, D., Stewart, D. J. (2003) Protective role of angiopoietin-1 in experimental pulmonary hypertension. Circ. Res. 92,984-991Epub 2003 Apr 2010
- ↵
Zhao, Y. D., Courtman, D. W., Ng, D. S., Robb, M. J., Deng, Y. P., Trogadis, J., Han, R. N., Stewart, D. J. (2006) Microvascular regeneration in established pulmonary hypertension by angiogenic gene transfer. Am. J. Respir. Cell Mol. Biol.16,16
- ↵
- ↵
- ↵
Ohlfest, J. R., Frandsen, J. L., Fritz, S., Lobitz, P. D., Perkinson, S. G., Clark, K. J., Nelsestuen, G., Key, N. S., McIvor, R. S., Hackett, P. B., Largaespada, D. A. (2005) Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. Blood 105,2691-2698Epub 2004 Dec 2692
- ↵
Liu, L., Mah, C., Fletcher, B. S. (2006) Sustained FVIII expression and phenotypic correction of hemophilia a in neonatal mice using an endothelial-targeted Sleeping Beauty transposon. Mol. Ther. 3,3
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
Patel, J. M., Zhang, J., Block, E. R. (1996) Nitric oxide-induced inhibition of lung endothelial cell nitric oxide synthase via interaction with allosteric thiols: role of thioredoxin in regulation of catalytic activity. Am. J. Respir. Cell Mol. Biol. 15,410-419
- ↵
- ↵
Guignabert, C., Raffestin, B., Benferhat, R., Raoul, W., Zadigue, P., Rideau, D., Hamon, M., Adnot, S., Eddahibi, S. (2005) Serotonin transporter inhibition prevents and reverses monocrotaline-induced pulmonary hypertension in rats. Circulation111,2812-2819
- ↵
Dupuy, A. J., Clark, K., Carlson, C. M., Fritz, S., Davidson, A. E., Markley, K. M., Finley, K., Fletcher, C. F., Ekker, S. C., Hackett, P. B., et al (2002) Mammalian germ-line transgenesis by transposition. Proc. Natl. Acad. Sci. U. S. A.99,4495-4499Epub 2002 Mar 4419
- ↵
- ↵
Ichinose, F., Roberts, J. D., Jr, Zapol, W. M. (2004) Inhaled nitric oxide: a selective pulmonary vasodilator: current uses and therapeutic potential. Circulation109,3106-3111
- ↵
Miller, O. I., Tang, S. F., Keech, A., Celermajer, D. S. (1995) Rebound pulmonary hypertension on withdrawal from inhaled nitric oxide. Lancet 346,51-52
- ↵
- ↵
Galie, N., Manes, A., Branzi, A. (2003) Prostanoids for pulmonary arterial hypertension. Am. J. Respir. Med. 2,123-137
- ↵
- ↵
- ↵
Chen, L., Daum, G., Forough, R., Clowes, M., Walter, U., Clowes, A. W. (1998) Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery. Circ. Res. 82,862-870
- ↵
- ↵
Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat, N., McIntyre, E., Radford, I., Villeval, J. L., Fraser, C. C., Cavazzana-Calvo, M., Fischer, A. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348,255-256
- ↵
Yant, S. R., Wu, X., Huang, Y., Garrison, B., Burgess, S. M., Kay, M. A. (2005) High-resolution genome-wide mapping of transposon integration in mammals. Mol. Cell Biol. 25,2085-2094
- ↵
Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., Calos, M. P. (2001) Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol. Cell Biol. 21,3926-3934
- ↵
Ohana, P., Gofrit, O., S, A., Al-Sharef, W., Mizrahi, A., Birman, T., Schneider, T., Matouk, I., de Groot, N., Tavdy, E., Ami Sidi, A., Hochberg, A. (2004) Regulatory sequences of the H19 gene in DNA based therapy of bladder cancer.Gene Ther. Mol. Biol. 8,181-192
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http://www.fasebj.org/cgi/doi/10.1096/fj.06-6254fje
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