- Email: [email protected]
Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in Rabbits
Fengwei Zhang, MD, PhD, Shuming Wu, MD, Xianshuo Lu, MD, Mo Wang, MD, and Meiming Liu, MD Department of Cardiovascular Surgery, Qilu Hospital of Shandong University, Jinan, Shandong Province, China
Background. Nitric oxide, a potent vasodilator with an important role in the regulation of pulmonary vascular tone, is synthesized by a family of nitric oxide synthases. To determine whether endothelial nitric oxide synthase (eNOS) gene transfer may prevent pulmonary hypertension, the effects of transfer of the eNOS gene to the lung were studied in rabbits with pulmonary hypertension induced by high pulmonary blood flow. Methods. Adenoviral vector encoding the eNOS gene was intratracheally transfected into the lung of rabbits with flow-induced pulmonary hypertension. Rabbits instilled intratracheally with adenoviral vector without encoding the eNOS gene served as a control group. Hemodynamic data were recorded before and after transfection, and transgene expression was investigated. Results. Pulmonary hypertension was significantly attenuated in eNOS gene–transfected rabbits compared with control animals (mean pulmonary arterial pressure, 22.3 ⴞ 5.5 versus 41.0 ⴞ 6.9 mm Hg; pulmonary vascular
resistance, 326 ⴞ 42 versus 618 ⴞ 66 dynes · s · cmⴚ5; p < 0.01). Systemic arterial pressure and systemic vascular resistance were unaffected. There was an increase in calcium-dependent conversion of L-arginine to L-citrulline in the lung (16.81 ⴞ 0.72 versus 4.11 ⴞ 0.41 pmol · mg proteinⴚ1 · hⴚ1) and cyclic guanosine monophosphate levels (0.138 ⴞ 0.015 versus 0.065 ⴞ 0.003 pmol/mg protein). Immunohistochemical staining showed expression of the eNOS gene was detected mainly in endothelial cells of small pulmonary vessels. Transgene expression was confirmed using Western blot analysis. Conclusions. These data suggest that intratracheal adenoviral-mediated eNOS gene transfer can attenuate flow-induced pulmonary hypertension in rabbits and may represent a new form of therapy for the treatment of flow-induced pulmonary hypertension.
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constriction [2, 3]. It is possible therefore that transfer of the eNOS gene to the lung may correct PH secondary to increased pulmonary blood flow. The present study was undertaken to investigate whether intratracheal adenoviral gene transfer of eNOS ameliorates PH induced by arteriovenous shunt.
espite recent advances in medical and surgical treatment, pulmonary hypertension (PH) secondary to congenital heart disease is still a serious problem that leads to high morbidity and mortality postoperatively. The available treatment options use vasorelaxants such as continuously infused prostaglandin analogs or inhaled nitric oxide (NO); however, they are still far from a curative treatment. More effective and less invasive therapy based on the pathophysiology of PH should thus be developed. Nitric oxide synthesized by endothelial nitric oxide synthase (eNOS) is a potent vasodilator and is considered to play an important role in regulating pulmonary vascular tone. Gene transfer of eNOS for cardiovascular diseases is an attractive new approach and may produce prolonged attenuation of PH. Previous studies showed rat smooth muscle cells transfected with eNOS under the control of the cytomegalovirus (CMV) enhancer– promoter could treat rats with monocrotaline-induced PH [1]. Adenoviral transfer of the eNOS gene to the lung of the mouse and rat reduced hypoxic pulmonary vasoAccepted for publication Aug 21, 2007. Address correspondence to Dr Zhang, Department of Cardiac Surgery, Linyi People’s Hospital, Linyi 276003, Shandong Province, China; e-mail: [email protected].
© 2008 by The Society of Thoracic Surgeons Published by Elsevier Inc
(Ann Thorac Surg 2008;85:581– 6) © 2008 by The Society of Thoracic Surgeons
Material and Methods Recombinant Adenovirus Vectors Construction, propagation, purification, and evaluation of recombinant adenovirus encoding the eNOS gene (AdCMVeNOS) used in the present study were described previously [4]. Adenovirus without encoding the eNOS gene (AdCMV-Null) was used as a control vector. Recombinant adenoviruses were plaque-purified, and virus titer was determined by plaque assay on 293 cells in culture. After purification the virus was suspended in phosphate-buffered saline solution (pH 7.4) with 3% sucrose and stored at ⫺80°C until use.
Animals Forty male New Zealand white rabbits weighing 2.0 to 2.8 kg were used in this study. Animal care and procedures 0003-4975/08/$34.00 doi:10.1016/j.athoracsur.2007.08.043
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Table 1. Influence of Transfection With Adenoviral Vector Encoding the Endothelial Nitric Oxide Synthase Gene and With Null Adenovirus on Hemodynamic Values in Rabbits With Pulmonary Hypertension Pretransfection CARDIOVASCULAR
Variable mPAP (mm Hg) mSAP (mm Hg) CO (mL · min⫺1) PVR (dyne · s · cm⫺5) SVR (dyne · s · cm⫺5) a
Posttransfection
AdCMVeNOS
AdCMV-Null
AdCMVeNOS
AdCMV-Null
38.9 ⫾ 8.8 82.0 ⫾ 7.1 202.5 ⫾ 24.8 612 ⫾ 55 1,568 ⫾ 135
40.5 ⫾ 9.7 82.0 ⫾ 6.9 209.5 ⫾ 31.2 633 ⫾ 38 1,590 ⫾ 123
22.3 ⫾ 5.5a 81.9 ⫾ 7.1 218.8 ⫾ 19.6 326 ⫾ 42a 1,612 ⫾ 136
41.0 ⫾ 6.9 82.2 ⫾ 7.5 205.5 ⫾ 21.1 618 ⫾ 66 1,567 ⫾ 155
p ⬍ 0.01 when compared with AdCMV-Null.
CO ⫽ cardiac output; mPAP ⫽ mean pulmonary arterial pressure; resistance; SVR ⫽ systemic vascular resistance.
were in accordance with the guidelines specified by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experimental Center of Shandong University. Rabbits underwent a right common carotid artery anastomosis to the external jugular vein, followed by proximal external jugular vein ligation. An arteriovenous shunt was created to mimic the chronically increased pulmonary artery flow state of a congenital heart defect [5]. The pressure change of the pulmonary artery was recorded before the shunt operation and 3 months after the shunt operation. Twelve rabbits died during the 3-month interval after shunt surgery, yielding a survival rate of 70%. A shunt patency rate of 78.5% was seen in 22 rabbits. The criteria of PH were met in 18 rabbits with mean pulmonary arterial pressure of 39.9 ⫾ 8.7 mm Hg. These rabbits were randomly assigned to receive intratracheal instillation of either the AdCMVeNOS (n ⫽ 9) or AdCMV-Null (n ⫽ 9). The rabbits were anesthetized with pentobarbital (30 mg/kg intravenously), intubated with an endotracheal tube (internal diameter, 3.5 mm), and mechanically ventilated (Harvard KTR-4, NatureGene Corp, Medford, NJ). A small silicone elastomer catheter was introduced in the trachea distal to the endotracheal tube. Viral solution (1 mL containing 5 ⫻ 109 plaque-forming units of AdCMVeNOS or AdCMV-Null) was instilled into the trachea for 10 minutes.
mSAP ⫽ mean systemic arterial pressure;
PVR ⫽ pulmonary vascular
medical Engineering, Chinese Academy of Medical Sciences, Tianjin, China), and systemic and pulmonary vascular resistances were calculated.
Endothelial Nitric Oxide Synthase Activity Lung samples were homogenized in a solution of 250 mmol/L Tris · HCl (pH 7.4), 10 nmol/L EDTA, and 10 mmol/L ethylene glycol-bis(-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) and centrifuged at 13,800 Gal for 10 minutes at ⫹4°C. The supernatant was incubated in a solution of 10 mmol/L reduced nicotinamide-adenine dinucleotide phosphate, 1 Ci/L (1 Ci ⫽ 37 GBq) l-[3H] arginine, 6 mmol/L CaCl2, 50 mmol/L Tris · HCl (pH 7.4), 6 mol/L tetrahydrobiopterin, 2 mol/L flavin adenine dinucleotide, and 2 mol/L flavin mononucleotide for 60 minutes at 24°C. The reaction was stopped with stop buffer (2 mmol/L EGTA, 2 mmol/L EDTA, and 20 mmol/L Hepes buffer, pH 5.5). The radioactivity of the sample eluate was measured by liquid scintillation counting. Enzyme activity was expressed as
Hemodynamic Measurements Rabbits were anesthetized, intubated, and mechanically ventilated. Oxygen concentration was maintained at 40%. A femoral artery was cannulated (PE-10 tubing, outer diameter, 0.6 mm, inner diameter, 0.3 mm; Shinetech Instruments Co, Ltd, Taibei, Taiwan) for measurement of systemic arterial pressure. A designed single-lumen catheter (PE-10 tubing) was constructed with a curved tip to facilitate passage through the right heart. The catheter was passed through the right femoral vein and manipulated through the right ventricle into the pulmonary artery. Mean pulmonary arterial pressure and systolic pulmonary arterial pressure were measured simultaneously (RM 6000 physiological polygraph; Nihon Kohden Corp, Tokyo, Japan). Cardiac output was measured by impedance plethysmography (XL-200; Institute of Bio-
Fig 1. Comparison of calcium-dependent l-[3H] arginine conversion to l-[3H] citrulline in rabbit lung tissue 4 days after transfection with adenovirus encoding the endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). Values are picomoles per milligram of protein per hour of citrulline formation. *p ⬍ 0.01, when compared with the control group (without the endothelial nitric oxide synthase gene).
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antibody (Jinmei Biotech Co, Shenzhen, China) for 60 minutes. The sections were rinsed with phosphatebuffered saline solution and incubated with secondary antibody for 30 minutes, then incubated in avidin-biotin complex for 30 minutes at room temperature. Diaminobenzidine and hydrogen peroxide were used for development. The slides were lightly counterstained with hematoxylin and then dehydrated sequentially.
Western Blotting
Fig 2. Bar graphs showing the tissue concentrations of cyclic guanosine monophosphate (cGMP) in rabbit lung transfected with adenovirus encoding the endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). *p ⬍ 0.01, when compared with the control group (without the endothelial nitric oxide synthase gene).
citrulline production in picomoles per milligram of protein per hour.
Cyclic Guanosine Monophosphate Levels Four days after transfection, lungs tissue were quickfrozen in liquid nitrogen and stored at ⫺70°C until cyclic guanosine monophosphate (cGMP) levels were measured. Whole lung tissue was homogenized and centrifuged for 5 minutes at 4°C, and the supernatant was transferred to a 10-mL test tube. The samples were assayed for cGMP by using a direct immunoassay kit (Biovision Inc, CA). Lung cGMP levels are expressed as picomoles per milligram of protein.
Immunohistochemistry Rabbit lungs were fixed in phosphate-buffered 4% formalin. Sections were rehydrated and digested with proteinase K at room temperature, then washed with phosphate-buffered saline solution containing 2.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 138 mmol/L NaCl, and 8.1 mmol/L Na2HPO4. Endogenous peroxidase activity was reduced by immersion in 3% hydrogen peroxide in methanol. After rinsing, sections were covered in 10% goat serum for 30 minutes and incubated with eNOS
The lungs were removed and immediately frozen in liquid nitrogen. To extract total protein, lungs were homogenized in ice-cold buffer with NaCl and incubated on ice for 30 minutes. After centrifugation twice at 4°C for 20 minutes, the supernatant was mixed with an equal volume of 2% sodium dodecyl sulfate and fractionated using 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were then transferred to a nitrocellulose membrane by semidry electroblotting for 1 hour. The membranes were blocked in blocking reagent for 1 hour at room temperature and incubated with a primary monoclonal mouse anti-eNOS immunoglobin G antibody (Jinmei Biotech Co, Shenzhen, China). Bound antibody was detected with secondary antibody (Jinmei Biotech Co, Shenzhen, China) and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech Inc, Piscataway, NJ). Protein levels were quantified by scanning densitometry. Protein expression levels are given in arbitrary units.
Statistics All results are expressed as mean ⫾ standard deviation. The data were analyzed by using unpaired Student’s t tests. A probability value of less than 0.01 was used as the criterion for statistical significance.
Results Pulmonary Hemodynamics Hemodynamic variables in rabbits with PH after intratracheal administration of AdCMVeNOS or AdCMV-Null are presented in Table 1. Before adenovirus administration, mean pulmonary arterial pressure, mean systemic arterial pressure, cardiac output, pulmonary arterial resistance, and systemic vascular resistance were similar in animals receiving AdCMV-Null or AdCMVeNOS. Four Fig 3. (A) Intense endothelial nitric oxide synthase staining (arrow) was observed in vascular endothelium of small-sized pulmonary arteries of rabbits transfected with adenovirus encoding the endothelial nitric oxide synthase gene. (B) There was little endothelial nitric oxide synthase expression (arrow) in rabbits transfected with adenovirus without the gene. Magnification ⫻100.
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days after transfection, mean pulmonary arterial pressure and pulmonary vascular resistance were significantly reduced in animals transfected with AdCMVeNOS when compared with values in the AdCMV-Null group (p ⬍ 0.01).
Endothelial Nitric Oxide Synthase Activity The activity of the eNOS transgene was determined by measuring the conversion of l-[3H]arginine to l-[3H]citrulline in lung tissue from rabbits transfected with AdCMVeNOS and AdCMV-Null. Calcium-dependent l-[3H]arginine to l-[3H]citrulline was significantly lower in lung tissue from rabbits transfected with AdCMV-Null when compared with values in AdCMVeNOS rabbits (Fig 1).
Cyclic Guanosine Monophosphate Levels Data of cGMP levels measured in lung tissue transfected with AdCMVeNOS or AdCMV-Null show lung cGMP concentrations were significantly lower in rabbits transfected with AdCMV-Null when compared with values in rabbits transfected with AdCMVeNOS (Fig 2).
Immunohistochemistry Figure 3 illustrate the sites of eNOS expression. Lung eNOS protein is predominantly expressed in vascular endothelium of AdCMVeNOS-transfected rabbits, but not in AdCMV-Null–treated rabbits.
Fig 4. Western immunoblot analysis of endothelial nitric oxide synthase (eNOS) protein (135 kDa) levels in whole lung tissue of rabbit 4 days after transfection of adenovirus encoding endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). *p ⬍ 0.01. Statistical analysis was performed by comparing the density of bands quantified by scanning densitometry in the two groups.
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Western Blotting Western immunoblot analysis of whole lung tissue using a monoclonal antibody detected abundant levels of eNOS (135 kDa) protein in rabbit transfected with AdCMVeNOS; only very low levels were detected in lung extracts of AdCMV-Null–transfected rabbits (Fig 4).
Comment In the present study, recombinant adenovirus carrying the gene encoding eNOS (AdCMVeNOS) was instilled into rabbit lungs and its effect on PH induced by arteriovenous shunt was measured. Results show that adenovirally mediated transfer of the eNOS gene to the rabbit lungs decreased the elevated level of pulmonary arterial pressure and pulmonary vascular resistance associated with high pulmonary blood flow. At the same time, systemic vascular resistance was not altered. After AdCMVeNOS gene transfer, the eNOS is expressed in the endothelium of resistance-sized intrapulmonary arteries. Immunoblot studies demonstrated abundant levels of immunoreactive eNOS protein in lung extracts from the rabbits with intratracheal administration of AdCMVeNOS. Calciumdependent NO synthase activity and cGMP levels were significantly increased. These data suggest that transfer of the eNOS gene has activity in the lung of rabbits with flow-induced PH and can act as a selective pulmonary vasodilator without systemic side effects. The importance of the NO signaling pathway in the pathophysiology of PH is well recognized [6]. It is well established that NO has pulmonary vasodilator activity, and inhaled NO is beneficial in some forms of PH, particularly in pediatric patients [7–10]. Study in patients with primary and secondary PH reported reduced eNOS expression in pulmonary vascular endothelium. The lungs of patients with PH were found to contain significantly reduced eNOS immunoreactivity and eNOS messenger RNA and showed a significant inverse correlation between the level of immunoreactivity and the severity of the pathologic alterations in the pulmonary vasculature [11]. This has initiated interest in therapeutic strategies aimed at increasing NO and cGMP signaling. Studies in the rat and mouse show that adenovirally mediated eNOS gene transfer increases lung cGMP levels, blunts the response to ventilatory hypoxia, and reduces pulmonary vascular resistance in bleomycin-induced PH [2, 3]. Adenovirally mediated transfer of the eNOS gene to the lung in large part corrects a genetic deficiency resulting from eNOS deletion, suggesting a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced [12]. The mechanism of PH induced by high pulmonary blood flow is still unclear. Recent evidence suggests that eNOS gene expression was upregulated in the lungs of an animal model with flow-induced PH [13–16]. Increases in the expression of NO synthase do not necessarily imply increase in NO production, as the increased protein might have decreased enzyme activity [17]. Lam and colleagues [18] reported that the pulmonary artery of rats
with flow-induced PH expressed higher levels of eNOS, although concentrations of cGMP were reduced. This indicated that despite increased expression of eNOS, biologic activity of NO is decreased because endothelial function is impaired [18, 19]. The present results show that eNOS activity and lung cGMP concentrations were significantly increased after eNOS gene transfer to the lung, indicating eNOS transgene is biologically active in the lung of the rabbits with flow-induced PH. In conclusion, intratracheal transfer of eNOS gene to the lung decreases the elevated pulmonary vascular resistance without adverse effects on systemic hemodynamics in rabbits with flow-induced PH. The reduction of PH is associated with augmented pulmonary eNOS synthesis and elevated eNOS activity and lung cGMP levels. Despite these results, eNOS gene therapy for PH remains a future possibility rather than a clinical reality; however, the present study suggests that eNOS gene transfer represents a potential therapeutic approach to PH.
References 1. Campbell AI, Kuliszewski MA, Stewart DJ. Cell-based gene transfer to the pulmonary vasculature: endothelial nitric oxide synthase overexpression inhibits monocrotalineinduced pulmonary hypertension. Am J Respir Cell Mol Biol 1999;21:567–75. 2. Champion HC, Bivalacqua TJ, D’Souza FM, et al. 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 1999;84:1422–32. 3. Janssens SP, Bloch KD, Nong Z, Gerard RD, Zoldhelyi P, Collen D. Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats. J Clin Invest 1996;98:317–24. 4. Wu S, Wang X, Guo L, Zi J. Adenovirus mediated endothelial nitric oxide synthase gene transfer prevents restenosis of vein grafts. ASAIO J 2004;50:272–7. 5. Chou TF, Chen YS, Yu CC, Chien CT, Chen CF. Simple methods to elevate pulmonary arterial pressure by pre- and post-tricuspid shunts in rats. Chin J Physiol 2002;45:131–5.
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6. Michelakis ED. The Role of the NO Axis and its therapeutic implications in pulmonary arterial hypertension. Heart Failure Rev 2003;8:5–21. 7. Nelin LD, Hoffman GM. The use of inhaled nitric oxide in a wide variety of clinical problems. Pediatr Clin North Am 1998;45:531– 48. 8. Finer NN, Barrington KJ. Nitric oxide for respiratory failure in infants born at or near term. Cochrane Database Syst Rev 2001;4:CD000399. 9. Kawakami H, Ichinose F. Inhaled nitric oxide in pediatric cardiac surgery. Int Anesthesiol Clin 2004;42:93–100. 10. Kinsella JP, Abman SH. Inhaled nitric oxide therapy in children. Paediatr Respir Rev 2005;6:190 – 8. 11. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214 –21. 12. Champion HC, Bivalacqua TJ, Greenberg SS, Giles TD, Hyman AL, Kadowitz PJ. Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice. Proc Natl Acad Sci USA 2002;99:13248 –53. 13. Black SM, Fineman JR, Steinhorn RH, Bristow J, Soifer SJ. Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol 1998;275:1643–51. 14. Jeon BH, Chang SJ, Hong YM, Yoon SY, Choe IS. Effect of high blood flow on the expression of endothelial constitutive nitric oxide synthase in rats with femoral arteriovenous shunts. Endothelium 2000;7:243–52. 15. Chou T-F, Wu M-S, Chien C-T, Yu C-C, Chen C-F. Enhanced expression of nitric oxide synthase in the early stage after increased pulmonary blood flow in rats. Eur J Cardiothorac Surg 2002;21:331– 6. 16. Qi J, Du J, Tang X, Li J, Wei B, Tang C. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary hypertension and vascular diseases in rats produced by aortocaval shunting. Heart Vessels 2004;19: 81– 8. 17. Rengasamy A, Johns RA. Determination of Km for oxygen of nitric oxide synthase isoforms. J Pharmacol Exp Ther 1996; 276:30 –3. 18. Lam CF, Peterson TE, Croatt AJ, Nath KA, Katusic ZS. Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2005;289:2334 – 41. 19. Nava E, Farre AL, Moreno C. Alterations to the nitric oxide pathway in the spontaneously hypertensive rat. J Hypertens 1998;16:609 –15.
INVITED COMMENTARY Pulmonary hypertension, regardless of the cause, represents a significant challenge to cardiothoracic surgeons with regard to preoperative risk assessment, perioperative management, and postoperative outcomes. Despite multiple pharmacologic strategies, including prostanoids, endothelin-receptor antagonists, and phosphodiesterase inhibitors, pulmonary hypertension remains a source of significant morbidity and mortality. One of the most recognized regulators of pulmonary vascular vasomotor tone is nitric oxide (NO). Otherwise known as endothelial-derived relaxation factor, NO is biosynthesized from L-arginine in the vasculature by two forms of nitric oxide synthase: constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Upregulation of endogenous NO or exogenous delivery of NO has been well studied in a variety of cardiovascular models as well © 2008 by The Society of Thoracic Surgeons Published by Elsevier Inc
as humans. Indeed adenoviral gene transfer of eNOS has been previously reported to ameliorate monocrotaline and hypoxia-induced pulmonary hypertension. Zhang and colleagues [1] report a therapeuticallyaimed study on the effects of tracheal-delivered adenoviral eNOS gene transfer in a rabbit model of pulmonary overcirculation. They establish a background associated with congenital heart disease by performing pretricuspid shunts (carotid-jugular bypass). They demonstrate successful delivery of the gene and achieve a functional response by significantly decreasing pulmonary vascular resistance. The model seems challenging as only 18 of 40 animals survived or developed significant pulmonary hypertension. Although their biochemical data is firm, some of the kinetics associated with gene transfer remains nebulous. Quite frankly, the effects of only 0003-4975/08/$34.00 doi:10.1016/j.athoracsur.2007.09.025
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Background. Nitric oxide, a potent vasodilator with an important role in the regulation of pulmonary vascular tone, is synthesized by a family of nitric oxide synthases. To determine whether endothelial nitric oxide synthase (eNOS) gene transfer may prevent pulmonary hypertension, the effects of transfer of the eNOS gene to the lung were studied in rabbits with pulmonary hypertension induced by high pulmonary blood flow. Methods. Adenoviral vector encoding the eNOS gene was intratracheally transfected into the lung of rabbits with flow-induced pulmonary hypertension. Rabbits instilled intratracheally with adenoviral vector without encoding the eNOS gene served as a control group. Hemodynamic data were recorded before and after transfection, and transgene expression was investigated. Results. Pulmonary hypertension was significantly attenuated in eNOS gene–transfected rabbits compared with control animals (mean pulmonary arterial pressure, 22.3 ⴞ 5.5 versus 41.0 ⴞ 6.9 mm Hg; pulmonary vascular
resistance, 326 ⴞ 42 versus 618 ⴞ 66 dynes · s · cmⴚ5; p < 0.01). Systemic arterial pressure and systemic vascular resistance were unaffected. There was an increase in calcium-dependent conversion of L-arginine to L-citrulline in the lung (16.81 ⴞ 0.72 versus 4.11 ⴞ 0.41 pmol · mg proteinⴚ1 · hⴚ1) and cyclic guanosine monophosphate levels (0.138 ⴞ 0.015 versus 0.065 ⴞ 0.003 pmol/mg protein). Immunohistochemical staining showed expression of the eNOS gene was detected mainly in endothelial cells of small pulmonary vessels. Transgene expression was confirmed using Western blot analysis. Conclusions. These data suggest that intratracheal adenoviral-mediated eNOS gene transfer can attenuate flow-induced pulmonary hypertension in rabbits and may represent a new form of therapy for the treatment of flow-induced pulmonary hypertension.
D
constriction [2, 3]. It is possible therefore that transfer of the eNOS gene to the lung may correct PH secondary to increased pulmonary blood flow. The present study was undertaken to investigate whether intratracheal adenoviral gene transfer of eNOS ameliorates PH induced by arteriovenous shunt.
espite recent advances in medical and surgical treatment, pulmonary hypertension (PH) secondary to congenital heart disease is still a serious problem that leads to high morbidity and mortality postoperatively. The available treatment options use vasorelaxants such as continuously infused prostaglandin analogs or inhaled nitric oxide (NO); however, they are still far from a curative treatment. More effective and less invasive therapy based on the pathophysiology of PH should thus be developed. Nitric oxide synthesized by endothelial nitric oxide synthase (eNOS) is a potent vasodilator and is considered to play an important role in regulating pulmonary vascular tone. Gene transfer of eNOS for cardiovascular diseases is an attractive new approach and may produce prolonged attenuation of PH. Previous studies showed rat smooth muscle cells transfected with eNOS under the control of the cytomegalovirus (CMV) enhancer– promoter could treat rats with monocrotaline-induced PH [1]. Adenoviral transfer of the eNOS gene to the lung of the mouse and rat reduced hypoxic pulmonary vasoAccepted for publication Aug 21, 2007. Address correspondence to Dr Zhang, Department of Cardiac Surgery, Linyi People’s Hospital, Linyi 276003, Shandong Province, China; e-mail: [email protected].
© 2008 by The Society of Thoracic Surgeons Published by Elsevier Inc
(Ann Thorac Surg 2008;85:581– 6) © 2008 by The Society of Thoracic Surgeons
Material and Methods Recombinant Adenovirus Vectors Construction, propagation, purification, and evaluation of recombinant adenovirus encoding the eNOS gene (AdCMVeNOS) used in the present study were described previously [4]. Adenovirus without encoding the eNOS gene (AdCMV-Null) was used as a control vector. Recombinant adenoviruses were plaque-purified, and virus titer was determined by plaque assay on 293 cells in culture. After purification the virus was suspended in phosphate-buffered saline solution (pH 7.4) with 3% sucrose and stored at ⫺80°C until use.
Animals Forty male New Zealand white rabbits weighing 2.0 to 2.8 kg were used in this study. Animal care and procedures 0003-4975/08/$34.00 doi:10.1016/j.athoracsur.2007.08.043
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Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in Rabbits
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ZHANG ET AL eNOS GENE TRANSFER AND PULMONARY HYPERTENSION
Ann Thorac Surg 2008;85:581– 6
Table 1. Influence of Transfection With Adenoviral Vector Encoding the Endothelial Nitric Oxide Synthase Gene and With Null Adenovirus on Hemodynamic Values in Rabbits With Pulmonary Hypertension Pretransfection CARDIOVASCULAR
Variable mPAP (mm Hg) mSAP (mm Hg) CO (mL · min⫺1) PVR (dyne · s · cm⫺5) SVR (dyne · s · cm⫺5) a
Posttransfection
AdCMVeNOS
AdCMV-Null
AdCMVeNOS
AdCMV-Null
38.9 ⫾ 8.8 82.0 ⫾ 7.1 202.5 ⫾ 24.8 612 ⫾ 55 1,568 ⫾ 135
40.5 ⫾ 9.7 82.0 ⫾ 6.9 209.5 ⫾ 31.2 633 ⫾ 38 1,590 ⫾ 123
22.3 ⫾ 5.5a 81.9 ⫾ 7.1 218.8 ⫾ 19.6 326 ⫾ 42a 1,612 ⫾ 136
41.0 ⫾ 6.9 82.2 ⫾ 7.5 205.5 ⫾ 21.1 618 ⫾ 66 1,567 ⫾ 155
p ⬍ 0.01 when compared with AdCMV-Null.
CO ⫽ cardiac output; mPAP ⫽ mean pulmonary arterial pressure; resistance; SVR ⫽ systemic vascular resistance.
were in accordance with the guidelines specified by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experimental Center of Shandong University. Rabbits underwent a right common carotid artery anastomosis to the external jugular vein, followed by proximal external jugular vein ligation. An arteriovenous shunt was created to mimic the chronically increased pulmonary artery flow state of a congenital heart defect [5]. The pressure change of the pulmonary artery was recorded before the shunt operation and 3 months after the shunt operation. Twelve rabbits died during the 3-month interval after shunt surgery, yielding a survival rate of 70%. A shunt patency rate of 78.5% was seen in 22 rabbits. The criteria of PH were met in 18 rabbits with mean pulmonary arterial pressure of 39.9 ⫾ 8.7 mm Hg. These rabbits were randomly assigned to receive intratracheal instillation of either the AdCMVeNOS (n ⫽ 9) or AdCMV-Null (n ⫽ 9). The rabbits were anesthetized with pentobarbital (30 mg/kg intravenously), intubated with an endotracheal tube (internal diameter, 3.5 mm), and mechanically ventilated (Harvard KTR-4, NatureGene Corp, Medford, NJ). A small silicone elastomer catheter was introduced in the trachea distal to the endotracheal tube. Viral solution (1 mL containing 5 ⫻ 109 plaque-forming units of AdCMVeNOS or AdCMV-Null) was instilled into the trachea for 10 minutes.
mSAP ⫽ mean systemic arterial pressure;
PVR ⫽ pulmonary vascular
medical Engineering, Chinese Academy of Medical Sciences, Tianjin, China), and systemic and pulmonary vascular resistances were calculated.
Endothelial Nitric Oxide Synthase Activity Lung samples were homogenized in a solution of 250 mmol/L Tris · HCl (pH 7.4), 10 nmol/L EDTA, and 10 mmol/L ethylene glycol-bis(-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) and centrifuged at 13,800 Gal for 10 minutes at ⫹4°C. The supernatant was incubated in a solution of 10 mmol/L reduced nicotinamide-adenine dinucleotide phosphate, 1 Ci/L (1 Ci ⫽ 37 GBq) l-[3H] arginine, 6 mmol/L CaCl2, 50 mmol/L Tris · HCl (pH 7.4), 6 mol/L tetrahydrobiopterin, 2 mol/L flavin adenine dinucleotide, and 2 mol/L flavin mononucleotide for 60 minutes at 24°C. The reaction was stopped with stop buffer (2 mmol/L EGTA, 2 mmol/L EDTA, and 20 mmol/L Hepes buffer, pH 5.5). The radioactivity of the sample eluate was measured by liquid scintillation counting. Enzyme activity was expressed as
Hemodynamic Measurements Rabbits were anesthetized, intubated, and mechanically ventilated. Oxygen concentration was maintained at 40%. A femoral artery was cannulated (PE-10 tubing, outer diameter, 0.6 mm, inner diameter, 0.3 mm; Shinetech Instruments Co, Ltd, Taibei, Taiwan) for measurement of systemic arterial pressure. A designed single-lumen catheter (PE-10 tubing) was constructed with a curved tip to facilitate passage through the right heart. The catheter was passed through the right femoral vein and manipulated through the right ventricle into the pulmonary artery. Mean pulmonary arterial pressure and systolic pulmonary arterial pressure were measured simultaneously (RM 6000 physiological polygraph; Nihon Kohden Corp, Tokyo, Japan). Cardiac output was measured by impedance plethysmography (XL-200; Institute of Bio-
Fig 1. Comparison of calcium-dependent l-[3H] arginine conversion to l-[3H] citrulline in rabbit lung tissue 4 days after transfection with adenovirus encoding the endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). Values are picomoles per milligram of protein per hour of citrulline formation. *p ⬍ 0.01, when compared with the control group (without the endothelial nitric oxide synthase gene).
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antibody (Jinmei Biotech Co, Shenzhen, China) for 60 minutes. The sections were rinsed with phosphatebuffered saline solution and incubated with secondary antibody for 30 minutes, then incubated in avidin-biotin complex for 30 minutes at room temperature. Diaminobenzidine and hydrogen peroxide were used for development. The slides were lightly counterstained with hematoxylin and then dehydrated sequentially.
Western Blotting
Fig 2. Bar graphs showing the tissue concentrations of cyclic guanosine monophosphate (cGMP) in rabbit lung transfected with adenovirus encoding the endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). *p ⬍ 0.01, when compared with the control group (without the endothelial nitric oxide synthase gene).
citrulline production in picomoles per milligram of protein per hour.
Cyclic Guanosine Monophosphate Levels Four days after transfection, lungs tissue were quickfrozen in liquid nitrogen and stored at ⫺70°C until cyclic guanosine monophosphate (cGMP) levels were measured. Whole lung tissue was homogenized and centrifuged for 5 minutes at 4°C, and the supernatant was transferred to a 10-mL test tube. The samples were assayed for cGMP by using a direct immunoassay kit (Biovision Inc, CA). Lung cGMP levels are expressed as picomoles per milligram of protein.
Immunohistochemistry Rabbit lungs were fixed in phosphate-buffered 4% formalin. Sections were rehydrated and digested with proteinase K at room temperature, then washed with phosphate-buffered saline solution containing 2.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 138 mmol/L NaCl, and 8.1 mmol/L Na2HPO4. Endogenous peroxidase activity was reduced by immersion in 3% hydrogen peroxide in methanol. After rinsing, sections were covered in 10% goat serum for 30 minutes and incubated with eNOS
The lungs were removed and immediately frozen in liquid nitrogen. To extract total protein, lungs were homogenized in ice-cold buffer with NaCl and incubated on ice for 30 minutes. After centrifugation twice at 4°C for 20 minutes, the supernatant was mixed with an equal volume of 2% sodium dodecyl sulfate and fractionated using 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were then transferred to a nitrocellulose membrane by semidry electroblotting for 1 hour. The membranes were blocked in blocking reagent for 1 hour at room temperature and incubated with a primary monoclonal mouse anti-eNOS immunoglobin G antibody (Jinmei Biotech Co, Shenzhen, China). Bound antibody was detected with secondary antibody (Jinmei Biotech Co, Shenzhen, China) and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech Inc, Piscataway, NJ). Protein levels were quantified by scanning densitometry. Protein expression levels are given in arbitrary units.
Statistics All results are expressed as mean ⫾ standard deviation. The data were analyzed by using unpaired Student’s t tests. A probability value of less than 0.01 was used as the criterion for statistical significance.
Results Pulmonary Hemodynamics Hemodynamic variables in rabbits with PH after intratracheal administration of AdCMVeNOS or AdCMV-Null are presented in Table 1. Before adenovirus administration, mean pulmonary arterial pressure, mean systemic arterial pressure, cardiac output, pulmonary arterial resistance, and systemic vascular resistance were similar in animals receiving AdCMV-Null or AdCMVeNOS. Four Fig 3. (A) Intense endothelial nitric oxide synthase staining (arrow) was observed in vascular endothelium of small-sized pulmonary arteries of rabbits transfected with adenovirus encoding the endothelial nitric oxide synthase gene. (B) There was little endothelial nitric oxide synthase expression (arrow) in rabbits transfected with adenovirus without the gene. Magnification ⫻100.
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days after transfection, mean pulmonary arterial pressure and pulmonary vascular resistance were significantly reduced in animals transfected with AdCMVeNOS when compared with values in the AdCMV-Null group (p ⬍ 0.01).
Endothelial Nitric Oxide Synthase Activity The activity of the eNOS transgene was determined by measuring the conversion of l-[3H]arginine to l-[3H]citrulline in lung tissue from rabbits transfected with AdCMVeNOS and AdCMV-Null. Calcium-dependent l-[3H]arginine to l-[3H]citrulline was significantly lower in lung tissue from rabbits transfected with AdCMV-Null when compared with values in AdCMVeNOS rabbits (Fig 1).
Cyclic Guanosine Monophosphate Levels Data of cGMP levels measured in lung tissue transfected with AdCMVeNOS or AdCMV-Null show lung cGMP concentrations were significantly lower in rabbits transfected with AdCMV-Null when compared with values in rabbits transfected with AdCMVeNOS (Fig 2).
Immunohistochemistry Figure 3 illustrate the sites of eNOS expression. Lung eNOS protein is predominantly expressed in vascular endothelium of AdCMVeNOS-transfected rabbits, but not in AdCMV-Null–treated rabbits.
Fig 4. Western immunoblot analysis of endothelial nitric oxide synthase (eNOS) protein (135 kDa) levels in whole lung tissue of rabbit 4 days after transfection of adenovirus encoding endothelial nitric oxide synthase gene (AdCMVeNOS) or with adenovirus without encoding the endothelial nitric oxide synthase gene (AdCMV-Null). *p ⬍ 0.01. Statistical analysis was performed by comparing the density of bands quantified by scanning densitometry in the two groups.
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Western Blotting Western immunoblot analysis of whole lung tissue using a monoclonal antibody detected abundant levels of eNOS (135 kDa) protein in rabbit transfected with AdCMVeNOS; only very low levels were detected in lung extracts of AdCMV-Null–transfected rabbits (Fig 4).
Comment In the present study, recombinant adenovirus carrying the gene encoding eNOS (AdCMVeNOS) was instilled into rabbit lungs and its effect on PH induced by arteriovenous shunt was measured. Results show that adenovirally mediated transfer of the eNOS gene to the rabbit lungs decreased the elevated level of pulmonary arterial pressure and pulmonary vascular resistance associated with high pulmonary blood flow. At the same time, systemic vascular resistance was not altered. After AdCMVeNOS gene transfer, the eNOS is expressed in the endothelium of resistance-sized intrapulmonary arteries. Immunoblot studies demonstrated abundant levels of immunoreactive eNOS protein in lung extracts from the rabbits with intratracheal administration of AdCMVeNOS. Calciumdependent NO synthase activity and cGMP levels were significantly increased. These data suggest that transfer of the eNOS gene has activity in the lung of rabbits with flow-induced PH and can act as a selective pulmonary vasodilator without systemic side effects. The importance of the NO signaling pathway in the pathophysiology of PH is well recognized [6]. It is well established that NO has pulmonary vasodilator activity, and inhaled NO is beneficial in some forms of PH, particularly in pediatric patients [7–10]. Study in patients with primary and secondary PH reported reduced eNOS expression in pulmonary vascular endothelium. The lungs of patients with PH were found to contain significantly reduced eNOS immunoreactivity and eNOS messenger RNA and showed a significant inverse correlation between the level of immunoreactivity and the severity of the pathologic alterations in the pulmonary vasculature [11]. This has initiated interest in therapeutic strategies aimed at increasing NO and cGMP signaling. Studies in the rat and mouse show that adenovirally mediated eNOS gene transfer increases lung cGMP levels, blunts the response to ventilatory hypoxia, and reduces pulmonary vascular resistance in bleomycin-induced PH [2, 3]. Adenovirally mediated transfer of the eNOS gene to the lung in large part corrects a genetic deficiency resulting from eNOS deletion, suggesting a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced [12]. The mechanism of PH induced by high pulmonary blood flow is still unclear. Recent evidence suggests that eNOS gene expression was upregulated in the lungs of an animal model with flow-induced PH [13–16]. Increases in the expression of NO synthase do not necessarily imply increase in NO production, as the increased protein might have decreased enzyme activity [17]. Lam and colleagues [18] reported that the pulmonary artery of rats
with flow-induced PH expressed higher levels of eNOS, although concentrations of cGMP were reduced. This indicated that despite increased expression of eNOS, biologic activity of NO is decreased because endothelial function is impaired [18, 19]. The present results show that eNOS activity and lung cGMP concentrations were significantly increased after eNOS gene transfer to the lung, indicating eNOS transgene is biologically active in the lung of the rabbits with flow-induced PH. In conclusion, intratracheal transfer of eNOS gene to the lung decreases the elevated pulmonary vascular resistance without adverse effects on systemic hemodynamics in rabbits with flow-induced PH. The reduction of PH is associated with augmented pulmonary eNOS synthesis and elevated eNOS activity and lung cGMP levels. Despite these results, eNOS gene therapy for PH remains a future possibility rather than a clinical reality; however, the present study suggests that eNOS gene transfer represents a potential therapeutic approach to PH.
References 1. Campbell AI, Kuliszewski MA, Stewart DJ. Cell-based gene transfer to the pulmonary vasculature: endothelial nitric oxide synthase overexpression inhibits monocrotalineinduced pulmonary hypertension. Am J Respir Cell Mol Biol 1999;21:567–75. 2. Champion HC, Bivalacqua TJ, D’Souza FM, et al. 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 1999;84:1422–32. 3. Janssens SP, Bloch KD, Nong Z, Gerard RD, Zoldhelyi P, Collen D. Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats. J Clin Invest 1996;98:317–24. 4. Wu S, Wang X, Guo L, Zi J. Adenovirus mediated endothelial nitric oxide synthase gene transfer prevents restenosis of vein grafts. ASAIO J 2004;50:272–7. 5. Chou TF, Chen YS, Yu CC, Chien CT, Chen CF. Simple methods to elevate pulmonary arterial pressure by pre- and post-tricuspid shunts in rats. Chin J Physiol 2002;45:131–5.
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6. Michelakis ED. The Role of the NO Axis and its therapeutic implications in pulmonary arterial hypertension. Heart Failure Rev 2003;8:5–21. 7. Nelin LD, Hoffman GM. The use of inhaled nitric oxide in a wide variety of clinical problems. Pediatr Clin North Am 1998;45:531– 48. 8. Finer NN, Barrington KJ. Nitric oxide for respiratory failure in infants born at or near term. Cochrane Database Syst Rev 2001;4:CD000399. 9. Kawakami H, Ichinose F. Inhaled nitric oxide in pediatric cardiac surgery. Int Anesthesiol Clin 2004;42:93–100. 10. Kinsella JP, Abman SH. Inhaled nitric oxide therapy in children. Paediatr Respir Rev 2005;6:190 – 8. 11. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214 –21. 12. Champion HC, Bivalacqua TJ, Greenberg SS, Giles TD, Hyman AL, Kadowitz PJ. Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice. Proc Natl Acad Sci USA 2002;99:13248 –53. 13. Black SM, Fineman JR, Steinhorn RH, Bristow J, Soifer SJ. Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol 1998;275:1643–51. 14. Jeon BH, Chang SJ, Hong YM, Yoon SY, Choe IS. Effect of high blood flow on the expression of endothelial constitutive nitric oxide synthase in rats with femoral arteriovenous shunts. Endothelium 2000;7:243–52. 15. Chou T-F, Wu M-S, Chien C-T, Yu C-C, Chen C-F. Enhanced expression of nitric oxide synthase in the early stage after increased pulmonary blood flow in rats. Eur J Cardiothorac Surg 2002;21:331– 6. 16. Qi J, Du J, Tang X, Li J, Wei B, Tang C. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary hypertension and vascular diseases in rats produced by aortocaval shunting. Heart Vessels 2004;19: 81– 8. 17. Rengasamy A, Johns RA. Determination of Km for oxygen of nitric oxide synthase isoforms. J Pharmacol Exp Ther 1996; 276:30 –3. 18. Lam CF, Peterson TE, Croatt AJ, Nath KA, Katusic ZS. Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2005;289:2334 – 41. 19. Nava E, Farre AL, Moreno C. Alterations to the nitric oxide pathway in the spontaneously hypertensive rat. J Hypertens 1998;16:609 –15.
INVITED COMMENTARY Pulmonary hypertension, regardless of the cause, represents a significant challenge to cardiothoracic surgeons with regard to preoperative risk assessment, perioperative management, and postoperative outcomes. Despite multiple pharmacologic strategies, including prostanoids, endothelin-receptor antagonists, and phosphodiesterase inhibitors, pulmonary hypertension remains a source of significant morbidity and mortality. One of the most recognized regulators of pulmonary vascular vasomotor tone is nitric oxide (NO). Otherwise known as endothelial-derived relaxation factor, NO is biosynthesized from L-arginine in the vasculature by two forms of nitric oxide synthase: constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Upregulation of endogenous NO or exogenous delivery of NO has been well studied in a variety of cardiovascular models as well © 2008 by The Society of Thoracic Surgeons Published by Elsevier Inc
as humans. Indeed adenoviral gene transfer of eNOS has been previously reported to ameliorate monocrotaline and hypoxia-induced pulmonary hypertension. Zhang and colleagues [1] report a therapeuticallyaimed study on the effects of tracheal-delivered adenoviral eNOS gene transfer in a rabbit model of pulmonary overcirculation. They establish a background associated with congenital heart disease by performing pretricuspid shunts (carotid-jugular bypass). They demonstrate successful delivery of the gene and achieve a functional response by significantly decreasing pulmonary vascular resistance. The model seems challenging as only 18 of 40 animals survived or developed significant pulmonary hypertension. Although their biochemical data is firm, some of the kinetics associated with gene transfer remains nebulous. Quite frankly, the effects of only 0003-4975/08/$34.00 doi:10.1016/j.athoracsur.2007.09.025
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