| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* 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
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
SPECIFIC AIMS
Primary pulmonary hypertension (PH) is a life-threatening disorder with limited treatment options. Gene therapy for PH is an alternative treatment strategy, yet viral vectors have inherent disadvantages, including immune activation. In the present study, we sought to investigate the efficacy of a nonviral transposon-based gene delivery system (Sleeping Beauty) to abrogate the PH that develops in response to monocrotaline (MCT). The endothelial nitric oxide synthase (eNOS) gene was chosen as an ideal candidate to test this approach, as deficiencies of this enzyme are thought to contribute to the pathogenesis of PH.
PRINCIPAL FINDINGS
1. eNOS-expressing transposon promotes the generation of NO in vitro and in vivo
A mutant phosphomimetic form of the human eNOS gene with constitutive enzymatic activity was cloned into the transposon vector pMSZ/cytomegalovirus (CMV), generating pMSZ/CMV-eNOS. Additional experiments were conducted to demonstrate the activity of this transposon expression vector. Western blotting of HUVECs transiently transfected with the eNOS transposon verified robust expression of the eNOS protein, with no immunoreactivity detected in control cells. To confirm biological activity, nitrite/nitrate concentrations and eNOS enzymatic activity were measured. 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. Culture medium from cells transiently transfected with the eNOS transposon contained high levels of NOx compared to the control cells (350 nM vs. 9 nM). To further verify eNOS enzymatic activity, assays were performed on membrane extracts 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).
To demonstrate that the transposon vector system can mediate long-term expression of eNOS in vivo, animals (male, Sprague-Dawley rats) received intravenous (i.v.) injections of the eNOS transposon complexed to linear polyethylenimine. 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. Western blotting revealed 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 a nonfunctional transposase (group 2). NOx measurements showed higher NOx levels present in lung homogenates from group 1 (18 nmol/mg protein), compared with that observed in group 2 (
5 nmol/mg protein). Splinkerette-mediated polymerase chain reaction (PCR) techniques were employed to show molecular evidence of transposon integration, which was observed only in animals receiving the active transposase. These results indicate that SB can mediate sustained eNOS expression within the lung, which is transposase-dependent.
2. Transposon-mediated gene therapy improves pulmonary hemodynamics and attenuates right ventricular hypertrophy
Sprague-Dawley rats were again injected with the eNOS transposon with (group 1) or without (group 2) functional transposase. Two days following gene delivery, the animals received a subcutaneous (s.c.) injection of MCT to promote pulmonary hypertension. Two control groups consisted of MCT alone-treated animals or nontreated controls. Four weeks later, animals from each group underwent cannulation of their pulmonary arteries to determine pulmonary artery blood pressures (PABP), and the hearts were excised and weighed to determine the right ventricle (RV) to whole heart (WH) wt ratios. The results demonstrated 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) or the MCT alone group (49.67±3.22 mmHg). 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), suggesting that 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). 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.
3. Transposon-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. 1
A) and MCT-treated animals (Fig. 1B
) demonstrate this phenomenon. Histological sections from eNOS transposon-treated animals showed reduced remodeling (Fig. 1C
, functional transposase; Fig. 2D
, mutant transposase). An independent pathologist blinded to the experimental design analyzed the lung histology and measured the wall thickness index in these sections. The results (Fig. 1E
) 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). These data illustrate that eNOS gene therapy can effectively attenuate pulmonary vascular remodeling.
|
|
CONCLUSIONS AND SIGNIFICANCE
Despite the diverse etiologies of pulmonary hypertension, the various disorders share similar histological and pathological findings, including the proliferation of endothelial and smooth muscle cells (SMCs) resulting in vascular remodeling, an inflammatory type reaction, and in situ thrombus formation with obliteration of distal arterioles. A schematic diagram of the molecules and pathways involved in the pathogenesis of PH is illustrated in Fig. 2
. To combat these processes, clinically relevant 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. While pharmacological agents can be effective, they have potential drawbacks such as the need for continuous i.v. infusion of prostacyclin derivatives or the use of nonselective vasodilators with potential side effects. These problems limit the therapeutic potential of these 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. 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. A growing body of literature points to the significant 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 (Fig. 2)
.
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. Although endothelial cells would be the ideal target, we chose to use a very active nonspecific promoter to obtain the highest possible level of eNOS expression within lung tissue. Using the CMV-driven eNOS transposon, we could demonstrate increased eNOS protein and NO production in vivo following gene transfer. Hemodynamic and histological data suggest that transposon-based eNOS expression prevented intimal wall hyperplasia and vascular remodeling (Fig. 1)
, reduced PABP, and attenuated right ventricular hypertrophy.
Although SB has been used in other animal paradigms, this is the first report of using SB-mediated gene delivery to treat PH. The 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. While SB is considered safer than retroviruses because of its random nature of integration, this problem could be addressed through the use of transposases with site-specific integration. Lastly, clinically relevant delivery methods of plasmid DNA are still needed. Although the polymer polyethylenimine has recently been used in humans, the efficiency of nonviral gene transfer could be improved through the synthesis more effective liposomes (e.g., cationic polymers and lipid) or lipoplexes. These complexes must be stable within plasma, transfect cells efficiently, and be able to navigate the cytoplasm to deliver the plasmid cargo to the nucleus. Given that few long-term treatment options are available for PH, other than lung transplantation, the success of this nonviral gene-based approach to attenuate the pathological processes driving PH warrants further investigations.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6254fje
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
