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(The FASEB Journal. 2006;20:2594-2596.)
© 2006 FASEB

Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats

Li Liu*, Hanzhong Liu{dagger}, Gary Visner{dagger} and Bradley S. Fletcher{ddagger},1

* Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida, USA;

{dagger} Division of Pulmonary Medicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; and

{ddagger} Medical Research Service, Department of Veteran Affairs Medical Center, Gainesville, Florida, USA

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{at}pharmacology.ufl.edu

ABSTRACT

Pulmonary hypertension (PH) is a life-threatening disorder with high mortality rates and limited treatment options. Gene therapy is an alternative treatment strategy, yet viral vectors have inherent disadvantages including immune activation. The Sleeping Beauty (SB) transposon is a nonviral method of gene delivery that overcomes some of these drawbacks. A SB-based transposon harboring a constitutively active endothelial nitric oxide synthase (eNOS) gene was administered to Sprague-Dawley rats via tail vein injection using the carrier polyethylenimine. Two days after transposon delivery, monocrotaline (MCT) was administered to induce PH. Hemodynamic, histological, and molecular measurements were performed four weeks later. Animals coinjected with transposase showed a significant reduction in pulmonary arterial pressure (PABP, 31.67±6.03 mmHg, P<0.01), an attenuation of right ventricle (RV) to whole heart (WH) wt ratios (0.227±0.0252, P<0.05) and a decrease in the pulmonary vessel wall thickness index (36.87%, P<0.001), compared with those animals receiving the eNOS transposon and a nonfunctional transposase (PABP 44.33±4.04 mmHg; RV/WH ratio 0.280±0.01; wall thickness index 62.14%) or control animals receiving MCT injection alone (PABP 49.67±3.22 mmHg; RV/WH ratio 0.290±0.0265; wall thickness index 71.99%). The physiological improvements correlated with therapeutic gene expression, suggesting that transposon-based genetic approaches have utility in the treatment of PH.—Liu, L., Liu, H., Visner, G., Fletcher, B. S. Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats.


Key Words: NOS • vasculature • remodeling • transposon

PRIMARY PULMONARY HYPERTENSION (PH) is a fatal disorder without intervention, and treatment options are few. The familial form of primary PH is a rare autosomal dominant disorder associated with mutations within the bone morphogenetic protein receptor-II (BMPR-II) gene (1) . However, in the majority of nonfamilial primary PH cases, the exact molecular defects precipitating the disease are unclear. Secondary PH is a syndrome common to a variety of lung and heart diseases, such as chronic obstructive lung disease, lung fibrosis, recurrent thromboembolism, or congenital heart defects (2) . Although the molecular mechanisms responsible for PH in the majority of cases remains unknown, recent studies indicate that endothelial cell dysfunction coupled with insufficient production of NO may promote the disorder (2 3 4) . In support of this hypothesis, decreased endothelial NOS (eNOS) expression has been observed in human PH patients (5) and in animal models of PH (6) . Furthermore, eNOS-deficient mice exhibit mild PH under normal atmospheric conditions yet have increased susceptibility to hypoxia-induced pulmonary hypertension (7) . Lastly, exogenous administration of NO significantly prevented the monocrotaline-induced remodeling of the pulmonary circulation in rats and reduced pulmonary artery pressures (8 , 9) . These data suggest that a reduction in resting pulmonary vascular NO synthesis could be involved in the etiology of PH.

Gene therapy represents an alternative method to currently available pharmacological treatments for PH. In fact, gene therapy for PH has been pursued for many years, mainly using adenoviral vectors and a vast array of different therapeutic genes (10 11 12 13 14) . Of these candidate genes, eNOS is attractive not only because of its ability to promote smooth muscle cell (SMC) relaxation via activation of soluble guanylate cyclase but also because of other properties, including the ability to inhibit SMC proliferation, migration, and matrix production (15) . NO also has been demonstrated to have antithombotic activity by preventing platelet aggregation and has antiinflammatory and immunoregulatory properties (15 , 16) . These observations suggest that the eNOS gene is an ideal target for genetic therapy of PH and its use in the studies described here are mainly to show the therapeutic potential of the gene delivery approach.

Although the adenoviral-based approaches for gene therapy in PH have shown success, these approaches have potential drawbacks of these and other viral vectors, such as the induction of inflammatory reactions and cellular toxicity that limit their utility (17 , 18) . In the case of pulmonary hypertension, the deleterious effects of an inflammatory immune reaction by the viral vector especially needs to be avoided due to the potential of accelerating the progression of PH. Gene therapy for PH that avoids the use of viral vectors has included plasmid-based (19 , 20) and cell-based (21 22 23) therapies. These nonviral gene transfer approaches have attracted increasing attention because of their reduced toxicity, simplicity of use, and lack of specific immune responses (24) . However, even these methodologies can be problematic, as cell-based therapies require significant ex vivo manipulation of syngeneic or autologous cells and plasmid-based therapies have limited therapeutic efficacy due to the lack of long-term transgene expression. This latter obstacle of plasmid-based therapies could be overcome through the use of integrating transposons.

The Sleeping Beauty (SB) transposon was the first characterized integrating transposon shown to have activity within mammalian cells (25) . Since its initial discovery, SB has proven to be an effective method to facilitate long-term expression of therapeutic genes following plasmid-based delivery within several animal models including hemophilia A (26 , 27) and B (28) , and murine tyrosinemia type I (29) . Long-term expression of the transgene is dependent on coexpression of the transposase and correlates with the presence of integrated transposons within the host genome. Depending on the in vivo techniques used to deliver plasmid-based therapies, specific organs or tissues can be transfected. For example, intravenous (i.v.) injection of DNA/PEI complexes has been previously shown to promote transgene expression primarily within the lung (30 , 31) and can be further targeted to lung endothelia through the use of cell-type specific promoters (32) . By utilizing physical techniques that tend to target lung tissue, we sought to determine whether a transposon-mediated approach could be useful for the treatment of pulmonary disorders, specifically PH. Following, in vivo delivery of SB transposons expressing the therapeutic gene eNOS, we could prevent some of the pathological consequences of monocrotaline-induced pulmonary hypertension in rats. This included a reduction in monocrotaline-induced pulmonary hypertension, an alleviation of RV hypertrophy, and reduction in the extent of pulmonary vascular remodeling. This is the first report describing in vivo delivery of transposon-based gene therapy to treat pulmonary hypertension, thereby providing new therapeutic strategies for the genetic treatment of this debilitating disorder.

MATERIALS AND METHODS

Plasmid construction and preparation
The full-length human eNOS cDNA (3.6 kb) harboring a phosphomimetic mutation (S1177D) [a gift from Dr. Dimmeler (33) ] was amplified by polymerase chain reaction (PCR) using primers containing NotI sites. The PCR product was TA cloned and subcloned into a transposon vector referred to as pMSZ/cytomegalovirus (CMV)-eNOS. This transposon is similar to pMSZ described previously (32) , except that the endothelin-1 promoter was replaced with a CpG-depleted CMV promoter obtained from the vector pGZB (34) . For these studies, a hyperactive transposase expression plasmid pTRUF-HSB16, as well as a nonfunctional transposase (pTRUF-mHSB), were used (35) . Plasmids for animal delivery were isolated by alkaline lysis and purified with an Endo-Free Giga Kit from Qiagen Inc. (Valencia, CA, USA)

Cell culture and transfection
Human umbilical vein endothelial cells (HUVEC) from American Type Culture Collection (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin/glutamine solution (Invitrogen). Cells were plated on 6 cm dishes and transfected with pMSZ/CMV-eNOS, or the control plasmid pMSZ/CMV-GFP, using Gene Jammer transfection reagent (Stratagene, La Jolla, CA, USA). Culture medium was collected for determination of NO production 48 h after transfection. The remaining cells were collected and fractionated for Western blotting and eNOS enzymatic activity.

Preparation of subcellular fractions
Subcellular fractions were prepared by washing cells twice in PBS and resuspending the pellet in 0.5 ml of HEPES buffer (HEPES 20 mM, pH 7.5; KCl 10 mM; MgCl2 1.5 mM; EDTA 1 mM; DTT 1 mM; and PMSF 0.1 mM) containing 250 mM sucrose and supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The cells were homogenized in a Dounce homogenizer and centrifuged twice at 750 g for 5 min at 4°C to collect nuclei and unbroken cells. The supernatants were centrifuged at 10,000 g for 1 h at 4°C to collect membrane pellets. Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit from Pierce Biotechnology (Rockford, IL, USA). The samples were kept at –80°C until assay.

Western blotting
Membrane fractions isolated from cells transfected with pMSZ/CMV-eNOS or a control plasmid (pMSZ/CMV-GFP), and lung tissues extracts from gene therapy or control animals were analyzed by Western blotting. Protein (20 µg) from cell fractions or 80 µg protein from lung extracts were separated by 7.5% SDS-PAGE under reducing conditions. Following transfer to PVDF membrane, protein was detected using a rabbit anti-human eNOS primary antibodies from BD Biosciences (San Jose, CA, USA; 1:3000 dilution), followed by incubation with polyclonal anti-rabbit HRP-conjugated secondary antibodies from DakoCytomation (Coppenhagen, Denmark; diluted to 1:2500). Bound primary antibody (Ab) was detected using a chemoluminescence kit from PerkinElmer Life Sciences (Wellesley, MA, USA). A prestained protein marker was used for molecular mass determinations. To confirm equal protein loading, membranes were stripped, washed, and reprobed with an Ab recognizing ß-actin.

NOS biological activity assay
For determination of NOS enzyme activity, the conversion of L-arginine to L-citrulline was assayed in membrane extracts as described as previously (36) . Briefly, the membrane fractions were incubated in 10 mM NADPH; L-(3H) arginine 1 µCi/liter (1 µCi=37 Gbq); 6 mM CaCl; 50 mM Tris HCl, pH 7.4; 6 µM tetrahydrobiopterin; 2 µM flavin adenine dinucleotide (FAD); 2 µM FMN for 60 min at 24°C. The reaction was stopped by the addition of 50 µM HEPES, pH 5.5, and 5 mM EDTA. The samples were run through columns containing the resin AG-50W-X8 (Bio-Rad, Hercules, CA, USA), which binds arginine, rinsed with ddH2O and collected into scintillation vials. Following the addition of Aquasol-II to the vials, the radioactivity was measured by liquid scintillation counting. Enzyme activity is expressed as citrulline production in pmol/min/mg protein.

Quantitative determination of NOx
NOx concentration in culture medium (expressed as nM) was measured with the 2-Channel Nitric Oxide Measuring System (Innovative Instruments, Inc., Wake Forest, NC, USA) according to the manufacturer’s instruction. NOx in lung tissue extracts from gene therapy-treated or control animals was measured using the Nitrate to Nitrite Reduction Kit and Griess Reaction Nitrite Kit from World Precision Instruments, Inc. (Sarasota, FL, USA) per manufacturer’s recommendation. Briefly, nitrate in lung extracts was first reduced to nitrite and then total nitrite concentrations measured using a colorimetric assay. Absorbance at 540 nm was measured using a microtiter plate reader. The concentration of NOx produced was determined based on standard curves produced at the same time. The final NOx concentration is expressed as pmol/mg protein.

Animals and in vivo gene delivery
Male Sprague-Dawley rats ({approx}200 g) were purchased from Harlan Sprague-Dawley. All rats were housed under conventional conditions and treated according to the NIH Guidelines for Animal Care with approval of the Institutional Animal Care and Use Committee of the University of Florida. Gene delivery was performed via tail vein injection in anesthetized rats using a DNA/PEI complex. Briefly, linear polyethylenimine (in vivo jetPEITM) was complexed with 450 µg of plasmid DNA in 5% glucose (Glc) (total injection vol 2 ml) using a charge ratio of 10. A 3:1 M ratio of transposon to transposase was used. Rats received either pMSZ/CMV-eNOS and a hyperactive transposase pTRUF-HSB16 (group 1) or pMSZ/CMV-eNOS and a nonfunctional transposase pTRUF-mHSB (group 2). Control and monocrotaline-only treated animals (MCT alone) received 2 ml of 5% Glc solution, which served as a negative control. Two days after gene delivery, rats received one dose of monocrotaline (60 mg/kg body wt) subcutaneously (s.c.).

Hemodynamic measurements
Four weeks after monocrotaline treatment, pulmonary artery blood pressure (PABP) was measured as has been described previously (37) . Briefly, animals were anesthetized with 5% isoflurane in oxygen, followed by intubation orotracheally with a 14-gauge TeflonTM angiocatheter and mechanically ventilated with a vol-controlled ventilator (Harvard Rodent Ventilator, Model 683) with 2% isoflurane in oxygen at a rate of 80 breaths/min, a tidal vol of 10 ml/kg, and an inspired fraction of oxygen of 1.0. A median laparo-sternotomy was performed and a Millar Micro-Tip@catheter (Millar Instruments, Inc., Houston, TX, USA) was inserted through the right ventricle into the pulmonary artery. The PABP was thus determined using a pressure transducer and PowerLab system (ADInstruments, Colorado Springs, CO, USA).

Histological and immunohistochemistric analysis
Immediately after completion of PABP measurements, lungs were flushed with ice-cold saline through the main pulmonary artery at a perfusion pressure of 20 cm H2O until white. The right lung was removed and snap-frozen in liquid nitrogen for biochemical studies. The left lung was perfused with 10% neutral buffered formalin, postfixed overnight, and separated into two parts. One part was embedded in paraffin wax, while the other part was quickly frozen in liquid nitrogen and stored at –80°C until sectioned for immunohistochemical staining. The heart was removed en bloc, and the right ventricular free wall was dissected. Right ventricular hypertrophy was evaluated by the calculation of wt ratio of right ventricular free wall to whole heart (RV/WH). Sections (5 µm) were prepared from paraffin-embedded lungs, and Verhoeff-van Gieson staining to highlight elastic lamina was performed for histological analysis. Pulmonary vascular remodeling was measured by calculating wall thickness index described elsewhere (38) . A total of 10–15 intra-acinar arteries from each rat was analyzed.

Integration sites identification
To identify the integration sites, a splinkerette PCR technique was used to recover sequences flanking transposon insertion site on the 3' side (right IR/DR element) using a technique previously described (39) . Potential clones were subjected to sequencing to identify integration sites, and basic local alignment search tool (BLAST) analysis was performed to determine the chromosomal location.

Statistics
Results are expressed as mean ± SEM unless indicated. P values were calculated using one-way ANOVA and the Newman-Keuls Multiple Comparison Test with values <0.05 considered statistically significant. All experiments had at least three animals per group unless stated otherwise.

RESULTS

In vitro analysis of eNOS expression by transposon vectors
A mutant phosphomimetic form of the human eNOS gene (S1177D) (33) with constitutive enzymatic activity was cloned into the unique NotI site within the transposon vector pMSZ/CMV, generating pMSZ/CMV-eNOS. A schematic representation of this plasmid as well as the transposase expression plasmid used in these studies are illustrated (Fig. 1 A). To verify that the vector can express eNOS protein with biological activity, HUVECs were transiently transfected with either the pMSZ/CMV-eNOS transposon, a green fluorescent protein expressing transposon pMSZ/CMV-GFP, or an empty vector; the latter two served as negative controls. Protein expression was verified by Western blotting of membrane fractions revealing an immunoreactive band with a molecular mass ~135 kDa from eNOS transposon-transfected cells, but no immunoreactivity from either of the control cells (Fig. 1B ). Biological activity was confirmed by measuring nitrite/nitrate concentrations and eNOS enzymatic activity. As NO is unstable and rapidly oxidized into other more stable products, such as nitrite and nitrate (NOx), the levels of these compounds can be used as indirect measures of NO production. As illustrated in Fig. 1C , the medium from cells transfected with the eNOS transposon contained high levels of NOx compared with control cells (350 nM vs. 9 nM). To further verify eNOS enzymatic activity, cells were harvested and fractionated into cytosolic and membrane fractions. Enzyme activity assays were performed measuring the conversion of [H3] L-arginine to L-citrulline. As expected, membrane fractions from cells transfected with the eNOS transposon had much higher activity than control fractions (4000 vs. 20 pmol/min/mg protein, P<0.001) (Fig. 1D ).


Figure 1
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  		Figure 1. The eNOS transposon and enzymatic activity analysis in vitro. A) Schematic representation of eNOS transposon and transposase expression vectors. A phosphomimetic form of the human eNOS gene (S1177D) was cloned into the transposon pMSZ, generating pMSZ/CMV-eNOS. Flanked by IR/DR elements, the eNOS gene is driven by a CpG-depleted CMV promoter. The transposase expression plasmid pTRUF-HSB16 encodes a mutant transposase with enhanced transposition activity, while the vector pTRUF-mHSB (not shown) encodes a nonfunctional transposase. B) In vitro production of eNOS protein by transposon plasmid. HUVECs transiently transfected with the indicated plasmids were harvested, and membrane fractions were separated by SDS-PAGE. As negative controls, membrane fractions were collected from pMSZ/CMV-empty and pMSZ/CMV-GFP transfected HUVECs. eNOS protein (135 kDa) was detected using standard Western blotting (arrow). Reprobing of the membrane with actin illustrates equivalent protein loading (arrowhead). C) NOx concentration in culture medium. HUVECs were transiently transfected with the eNOS transposon (pMSZ/CMV-eNOS) or control vectors (pMSZ-CMV-empty and pMSZ/cCMV-GFP). Culture medium was collected and NOx levels were quantified 48 h after transfection. The concentration is in nM, and results are expressed as mean ± SEM. ** P < 0.001. D) eNOS enzymatic activity in membrane fractions. Membrane fractions isolated in (B) were assayed for NOS activity by measuring the conversion of L-arginine to L-citrulline. The results are expressed as pmol/min/mg of protein plotting the mean, and SEM from three independent plates is graphed. ** P < 0.001.

Sleeping Beauty-mediated gene delivery and expression of eNOS in vivo
Animals received i.v. injection of eNOS transposon complexed with PEI, which facilitates transfection of lung (30 , 40) . Two days following gene delivery, animals received a s.c. injection of MCT to promote pulmonary hypertension. Four weeks later, lungs were excised, homogenized, and analyzed for eNOS protein expression. In addition, measurements of NOx production allow for an indirect assessment of NOS activity. The blots reveal robust expression of eNOS protein within the lungs of animals that received the pMSZ/CMV-eNOS transposon plus functional transposase (group 1), while lower levels of eNOS protein were detected in the lungs of animals receiving the eNOS transposon and the nonfunctional transposase (group 2). As the Ab is human-specific, no immunoreactive bands were observed in the lungs of control animals (Fig. 2 A). NOx measurements showed higher NOx levels present in lung homogenates from group 1 (P<0.001), compared with group 2 and MCT treatment alone (Fig. 2B ), although a small difference was found between groups 2 and MCT alone (P>0.5), which is likely from the contribution of nonintegrated extrachromosomal plasmid. Given that eNOS protein expression and NO production are reported to be decreased in monocrotaline-induced pulmonary hypertension (6) , we assume that the increased NOx concentrations observed are the result of eNOS gene delivery and transgene expression. These results indicate that SB can mediate sustained eNOS expression within the lung, which is transposase-dependent.


Figure 2
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  		Figure 2. In vivo eNOS protein and NOx detection in transposon-treated rats. A) Rats received plasmid DNA complexed to PEI via tail vein injection. Animals were split into three groups (n≥3/group): those receiving the eNOS transposon with active transposase (group 1), eNOS transposon and inactive transposase (group 2), or 5% dextrose as control (MCT alone). Two days after gene delivery, the animals received one dose of monocrotaline (60 mg/kg) s.c. Four weeks after monocrotaline treatment, lung tissues were harvested homogenized, and membrane fractions were subjected to Western blotting to detect eNOS protein. B) The NOx concentration in lung extracts from transposon-treated or control animals was measured using a Griess reaction nitrite/nitrate (NOx) kit. The concentration is expressed as nmol/mg protein, and results are expressed as mean ± SEM; **P < 0.001.

Transposon-mediated gene therapy improves pulmonary hemodynamics and attenuates right ventricular hypertrophy
The PABP and the RV to WH ratios were measured at 4 wk after monocrotaline injection in transposon-treated and control animals. PABP tracings were obtained from all animals in each experimental group, and the data are presented as the mean PABP ± the SEM. The results demonstrate a significant reduction of PABP in animals from group 1 (31.67±6.03 mmHg, P<0.01) compared with animals in group 2 (44.33±4.04 mmHg) and the MCT alone group (49.67±3.22 mmHg) (Fig. 3 A). Although a slight nonsignificant difference was found in the NOx production between group 2 and the MCT alone group (Fig. 2B ), less of a difference was found in their PABPs. The PABP in group 1 was still higher than that in normal rat (31.67±6.03 mmHg vs. 19.03±3.87 mmHg, P<0.01), which suggests the protection provided by increased eNOS activity was not complete. The RV to WH ratio had a similar pattern in that the ratio in group 1 was lower than that in group 2 and the MCT alone group (0.227±0.0252 vs. 0.280±0.01 and 0.290±0.0265, respectively, P<0.05). No significant difference was observed in the RV/WH when comparing group 1 with normal rats (0.227±0.0252 vs. 0.197±0.153, P>0.05) (Fig. 3B ). These results suggest that SB-mediated eNOS gene therapy can effectively reduce pulmonary hypertension induced by monocrotaline and reduce the pathological consequence, specifically right ventricular hypertrophy.


Figure 3
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  		Figure 3. Hemodynamic measurements and cardiac hypertrophy analysis. A) The mean PABP from control, MCT-treated, and transposon-treated animals following gene therapy and monocrotaline challenge is graphed for each group. B) Right ventricular hypertrophy was evaluated by calculation of the right ventricle to whole heart wt (RV/WH) ratio for each group. For each graph the mean ± SEM is reported, n = 3~4 and **P < 0.01 or *P < 0.05 for group 1 vs. MCT alone.

Sleeping Beauty-mediated eNOS gene therapy reduces pulmonary vascular remodeling
Pulmonary hypertension is associated with significant arterial wall hyperplasia and vascular remodeling. Representative histological sections of lung tissue from control (Fig. 4 A) and MCT-treated animals (Fig. 4B ) demonstrate this phenomenon. Histological sections from transposon-treated animals are also shown (Fig. 4C , eNOS and functional transposase; Fig. 4D , eNOS and mutant transposase). An independent pathologist blinded to the experimental design analyzed the lung histology. The results show a significant reduction in the wall thickness index in rats within group 1 compared to rats in group 2 or control rats receiving monocrotaline alone (36.7% vs. 61.7% and 72.1%, respectively, P<0.001). This data illustrate that eNOS gene therapy can effectively attenuate pulmonary vascular remodeling.


Figure 4
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  		Figure 4. Histological and morphological analysis. Representative images from each group are shown (A) Normal control; (B) MCT alone; (C) group 1; (D) group 2 (x400). E) Pulmonary vascular remodeling was evaluated by calculating the wall thickness index. Each value is mean ± SEM of 10–15 independent determinations from each lung; n = 3–4 animals and **P < 0.001 (group 1 vs. MCT alone).

Gene integration studies
Sustained transgene expression by SB-mediated gene delivery is presumably due to its ability to integrate the transgene into the target genome (28) . To demonstrate molecular evidence of transposon-mediated integration, a splinkerette-mediated PCR technique was employed (39) . Evidence of molecular integration was identified only in DNA from animals that received the functional transposase, suggesting that the observed differences in eNOS expression between animals in groups 1 and 2 were due to transgene integration allowing for long-term expression.

Genomic sequences present flanking the right side of the integrated transposon are presented in Table 1 .


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  		Table 1. Transposition integration sites identified by splinkerette PCR

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.

ACKNOWLEDGMENTS

This work was supported by a Scientist Development Grant from the American Heart Association (0430185N) and in part by a VA Medical Research Service award, both to B.S.F.

Received for publication May 18, 2006. Accepted for publication August 14, 2006.

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