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1Lung Research Laboratory, Royal Adelaide Hospital, 2Hanson Institute and 3University of Adelaide, Adelaide, South Australia, Australia; 4Department of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois; 5Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, Alabama; and 6Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge, United Kingdom
Submitted 14 January 2006 ; accepted in final form 26 January 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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hypoxia; growth substances; endothelium
superfamily of receptors (13). Decreased expression of BMPR2 has also been seen in secondary pulmonary hypertension, suggesting this pathway might also be important in the pathogenesis of a variety of common clinical situations in which pulmonary hypertension is a feature (1). The described BMPR2 mutations predict a downregulation of BMP signaling which, in turn, may lead to abnormal proliferative responses in pulmonary vascular cells. Ex vivo analysis of pulmonary smooth muscle cells from patients with familial PAH have confirmed abnormal proliferative responses to BMP ligands and TGF- (13). BMP signaling involves ligand-initiated heterodimerization of cell surface BMPR2 and BMPR1 (A or B); activation of the latter then phosphorylates a series of receptor-regulated (R-) SMAD proteins (e.g., Smad1, 5, and 8) that in turn complex with the common partner Smad (co-Smad, Smad4) and translocate into the nucleus to regulate target gene transcription (34). Dysfunctional SMAD signaling has been seen in PAH (35).
The importance of BMPR2 in pulmonary hypertension has recently been supported by transgenic mouse studies. West et al. (32) developed a mouse in which a dominant negative BMPR2 receptor is expressed under the control of a tetracycline-responsive, smooth muscle promoter cassette. When fed tetracycline, these mice developed pulmonary hypertension, and their propensity to do so was increased by mild hypoxia. Heterozygous BMPR2 knockout mice developed pulmonary hypertension in response to an inflammatory challenge mediated by intratracheal delivery of an adenoviral (Ad) vector carrying the gene for lipoxygenase, whereas genetically intact mice did not (24). Interestingly, however, these mice were relatively resistant to the pulmonary vascular remodeling effects of hypoxia (2), and a separate study has shown that mice deficient in one of the BMPR2 ligands, BMP4, were also resistant to hypoxia-induced pulmonary hypertension (4). Takahashi et al. (26) recently showed that hypoxia led to an early upregulation of the ligand BMP2, with a later downregulation of the BMPR2 receptor.
In the current study, we sought to further investigate the role of the BMPR2 pathway in the pathogenesis of pulmonary hypertension, and more specifically to determine whether this pathway could be exploited as a potential therapy. Thus, we developed Ad vectors containing the gene for wild-type BMPR2, validated the construct for functionality in vitro, and then determined the impact of vector administration on development of pulmonary hypertension and vascular remodeling in vivo. We used the rat model of hypoxic pulmonary hypertension in view of the indication from recent studies that interrelation between hypoxia and BMPR2 signaling exists (26, 32). For the in vivo studies, we have capitalized on our previously developed Ad vector targeting strategy whereby we link the Ad vector to a bispecific antibody that targets the virus to angiotensin-converting enzyme (ACE), a membrane-bound protease highly expressed on pulmonary endothelial cells. This strategy circumvents the relatively low levels of Ad receptors on pulmonary endothelium by providing an alternate means of viral attachment. The approach has been shown to substantially improve pulmonary vascular gene delivery and the effectiveness of vascular-directed gene therapy (12, 18, 20).
Herein we demonstrate that upregulation of the BMPR2 signaling pathway attenuates hypoxia-induced pulmonary hypertension in otherwise normal rats. These findings provide evidence for a role for the BMPR2 pathway beyond those cases directly related to genetic BMPR2 mutations. Importantly, the study demonstrates that upregulation of BMPR2 signaling could be developed as a potential therapeutic strategy for pulmonary hypertension, and, in principle, a gene therapy approach could be one means to achieve this.
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METHODS |
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Construction and validation of Fab-9B9 has previously been described (18, 20). Fab and MAb 9B9 were derivatized with the bifunctional crosslinker N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP; Pierce, Rockford, IL). SPDP was combined with 9B9 or Fab in PBS at a molar ratio of 4 SPDP:1 antibody and incubated shaking at room temperature (RT) for 30 min. The pH of Fab was lowered by adding 0.1 vol of 1 M sodium acetate, pH 4.5, and then the Fab was reduced by adding 1 mg of dithiothreitol (Bio-Rad, Hercules, CA). After a 5-min incubation, the reduced Fab was passed through a PD10 column (Pharmacia, Uppsala, Sweden), equilibrated in borate buffer, and then added immediately to the derivatized 9B9 and shaken at RT overnight. The conjugate mixture was purified by gel filtration (Superose 12 column; Pharmacia) in borate buffer, pH 8.5. Monomeric Fab and 9B9 were discarded, and fractions larger than 150 kDa were assessed for specificity.
Cells and cell cultures.
Normal mouse mammary gland epithelial cells (NMuMG), being constitutively competent for TGF- superfamily signaling (17), were used for signal transduction experiments. NMuMG were routinely cultured in DMEM (Gibco Invitrogen, Auckland, New Zealand) supplemented with 10% FCS (TRACE Scientific, Melbourne, Australia), 10 µg/ml insulin, 100 U/ml penicillin, and 50 µg/ml streptomycin. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in M199/Earle's balanced salts media (JRH Biosciences, Lenexa, KS) with 20% FCS, supplemented as described (5), and were used at passage 4 or less. Clonetics lung-derived normal human microvascular blood vessel endothelial cells (HMVEC-LBl) were cultured in EGM-2 MV as recommended by the supplier (Cambrex Bio Science, Walkersville, MD), supplemented with 5% FCS, and used between passages 4 and 9. All cells were grown in a 5% CO2 atmosphere at 37°C.
Animals. All animal protocols were reviewed and approved by the Animal Research Ethics Committee of the Institute of Medical and Veterinary Science (IMVS), Royal Adelaide Hospital, and The University of Adelaide. Experiments were conducted on 4-wk-old male Sprague-Dawley rats. The rats were housed in the IMVS animal care facility and fed food and water ad libitum.
Immunoblot for detecting BMPR2myc transgene expression. To assess the ability of our newly derived vector to achieve expression of BMPR2 receptors, 293T cells (2 x 105/well) were infected at a calculated dose of 200 plaque-forming units (pfu)/cell with either AdCMVBMPR2myc or control Ad vector (AdCMVLuc), as previously described (20). Forty-eight hours later, cell culture media was collected, and the transduced cells were lifted, washed, and lysed. Media or lysate samples (20 µl/lane) were loaded onto 12% polyacrylamide/SDS gels, electrophoresed, and then electroblotted to a Hybond-ECL membrane (Amersham Biosciences). After the electrotransfer, the membranes were blocked in 10% skim milk in Tris-buffered saline Tween 20 (TBS-T) and then incubated overnight at 4°C with rabbit anti-myc 1:2,500 (Sigma, St. Louis, MO). After being washed, the membrane was incubated with goat anti-rabbit horseradish peroxidase (1:50,000; Pierce Biotechnology), and reactive bands were detected by chemiluminescence (Amersham).
In vitro subcellular localization of BMPR2myc. Following overnight adherence in chamber slides (Nunc, Naperville, IL), NMuMG or HUVEC (4 x 104) were infected with Ad vectors at 100 pfu/cell. After 48 h, the cells were fixed using ice-cold methanol-5% acetone, blocked for 1 h in 3% goat serum, and then incubated for 1 h with a rabbit anti-myc antibody (1:500; Sigma) followed by detection using Alexa 594 goat anti-rabbit (1:500; Molecular Probes, Eugene, OR). Nuclei were counterstained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI; Roche, Mannheim, Germany) for 2 min. The sections were examined using an Olympus BX51 fluorescence microscope system (Japan); images were captured using a Photometrics CoolSnapfx camera (Roper Scientific, Tucson, AZ) and recorded using V++ Precision Imaging system (Digital Optics, Auckland, New Zealand).
Functional assessment of the delivered bmpr2myc gene. Functional analysis of delivered genes was assessed using an established model system (21). To detect activation of the BMP signaling pathway, we used a SMAD-luciferase reporter system. This system uses a plasmid (p3GC2wtLux) (9) containing a SMAD-sensitive promoter driving the expression of a luciferase reporter gene. NMuMG cells (1 x 105/well) were infected with Ad vector containing BMPR2myc gene (100 pfu/cell) or control Ad vector (AdCMVTKGFP), with each being cotransfected with 3 µg of plasmid/well (Effectene Transfection Reagent; Qiagen, Hilden, Germany). The following day, the medium was replaced with DMEM containing 0.1% FCS with or without 50 ng/ml of BMP-4 (R&D Systems, Minneapolis, MN). After a further 24 h, cells were lysed and assayed for luciferase activity using the Promega Luciferase Assay Reagent kit (Promega, Madison, WI) and a TD-20/20 luminometer (Turner BioSystems, Sunnyvale, CA) according to manufacturer protocols. The protein concentration of lysate was also determined using the Bio-Rad DC (detergent compatible) protein assay kit, and luciferase signals were then expressed as relative light units (RLU)/mg protein.
Proliferation assays. To determine the effect of BMPR2 transgene expression on cell proliferation, NMuMG (2 x 104/well) were infected (200 pfu/cells) with either AdCMVBMPR2myc or control Ad vector (AdCMVLuc). The following day, cells were quiesced in growth medium/0.1% FCS for 48 h. Following this, the medium was replaced with medium containing 1% FCS with or without 50 ng/ml of BMP-4 (R&D Systems). After 24 h, cell numbers were manually counted using a hemocytometer (American Optical, Buffalo, NY).
Apoptosis assay flow cytometry. Staining HMVEC-LBl with 7-amino-actinomycin D (7-AAD; Sigma) was used to quantify apoptosis. Briefly, HMVEC-LBl (2 x 105/well) were infected (200 pfu/cell) with either AdCMVBMPR2myc or control Ad vector (AdCMVLuc), and 24 h later cells were quiesced in growth medium/0.1% FCS for 24 h. Cells were then suspended by a brief trypsinization and washed twice in HEPES buffer, after which cells were resuspended in 100 µl of 2 µg/ml 7-AAD in HEPES buffer. One-hundred thousand events were acquired with a FACScalibur flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).
PCNA quantification by Western blot. Forty-eight hours after transfection, HMVEC-LBl (see above) were lysed, and 20–30 µg of total protein per sample was electrophoresed and transblotted as described above. After the electrotransfer, the membranes were blocked in 10% skim milk and then incubated overnight at 4°C with mouse anti-PCNA (clone PC10; Dako Cytometrics). Immunodetection was performed using ECL-Plus substrate (GE Healthcare), and densitometry measurements were made using a Typhoon 9410 Imager (Molecular Dynamics, Sunnyvale, CA). To confirm equal protein loading, blots were stripped with Restore Western stripping buffer (Pierce) and reprobed using an anti-actin antibody (Santa Cruz Biotechnology). Data quantification was performed using ImageQuant (Molecular Dynamics).
Immunodetection of BMPR2myc transgene expression in the pulmonary vasculature. Rats were injected with 5 x 109 pfu of either AdCMVBMPR2myc or AdCMVLuc complexed to Fab-9B9 via the lateral tail vein. Three days later, the animals were killed and lungs perfused via a 20G catheter (Insyte; BD, Sandy, UT) in the right ventricle and making a small slit in the left ventricle. The pulmonary vasculature was perfused with PBS/heparin (30 ml, 20 cmH2O) and then 30 ml of 10% neutral buffered formalin (ACE Chemical, Camden Park, SA, Australia). The lungs were inflated by tracheal instillation of formalin, and the trachea was tied off. After a 24-h fixation, the lung tissue was processed into paraffin blocks. Sections were cut at 5 µm and heat mounted (58°C for 1 h) on coated glass slides (Polysine, Menzel-Gläser). Lung sections were then deparaffinized and rehydrated through graded alcohols. Slides were immersed in preheated Target Retrieval Solution (DakoCytomation, Carpinteria, CA) at 95°C for 30 min, cooled, blocked in 10% goat serum, and incubated overnight at 4°C with a rabbit monoclonal anti-myc antibody (1:100; Sigma). The next day, washed slides were blocked with 10% goat serum followed by detection using Alexa 488 goat anti-rabbit (1:500; Molecular Probes). Nuclei were counterstained with DAPI. Slides were then mounted, and sections were examined using the fluorescent microscope system as described above.
Hypoxia studies. Male Sprague-Dawley rats were assigned to four groups (n = 6/group), three hypoxia-exposed groups and a normoxic control group (N). Rats in two of the hypoxic groups were given tail vein injection of Ads (5 x 109 pfu/rat), AdCMVTracLuc (an irrelevant viral control) and AdCMVBMPR2myc that had been targeted to deliver Ad vector to the pulmonary vascular endothelium (20). For this, each dose of Ad vector (5 x 109 pfu) was complexed with 5 µl of the Fab-9B9 antibody for 30 min at RT before injection. Injections were via the lateral tail vein using 27G insulin syringes (Terumo, Elkton, MD) with the total volume of adenovirus brought to 250 µl with sterile PBS. Twenty-four hours after injection, the hypoxic groups were exposed to normobaric hypoxia (10% O2) for 3 wk. Hypoxic exposures were achieved by placing the rats in a Plexiglas chamber and intermittently adding N2 to the chamber. Nitrogen gas flow was regulated by a solenoid valve controlled by an O2 analyzer (Edward Scientific, New South Wales, Australia). Carbon dioxide was removed by using soda lime granules, and excess humidity was prevented by refrigerated cooling of the recirculation circuit and anhydrous CaSO4 granules. Boric acid granules were used to keep NH3 levels within the chamber at a minimum. Normoxic rats were housed in identical cages while breathing room air.
Measurement of pulmonary hemodynamics. After 3 wk, the rats were anesthetized [ketamine (100 mg/kg ip) and xylazine (20 mg/kg ip)], and a small transverse cut was made in the proximal right external jugular vein through which an introducer and a PE10 catheter (Portex, Kent, UK) were passed. The introducer was a blunted 7.5-cm, 18G spinal needle (Sensi-Touch; Sherwood Medical, St. Louis, MO) with the tip turned up to 30°, which was tied securely with black braided silk suture (4/0 Ethicon; Johnson & Johnson, Sydney, Australia). The catheter was attached to a pressure transducer (Transpac IV, Abbott, Ireland) coupled to a polygraph (Neomedix). The catheter was then advanced into the pulmonary artery, and pressure was recorded. Finally, after a lethal injection of ketamine, the thorax was opened and lungs were perfused, inflated, and fixed as described above. The heart and lungs were excised en bloc. The heart was weighed, and the Fulton Index [ratio of right ventricular free wall weight over the sum of septum + left ventricular free wall weight (RV/S+LV)] was calculated.
Evaluation of vascular remodeling.
After 24 h of fixation, the lung tissue was processed with sections cut and heat mounted as above. Lung sections were then deparaffinized with xylene and taken to absolute alcohol followed by an endogenous peroxidase block with H2O2. Slides were immersed in preheated Target Retrieval Solution (DakoCytomation) at 95°C for 30 min, cooled, and blocked with 2.5% horse serum for 30 min. Sections then incubated overnight at RT with the primary antibody for smooth muscle 
-actin (Sigma) diluted 1:60,000 in 2.5% horse serum. The following day, slides were washed twice in PBS for 3 min followed by treatment using the Quick Kit (Vector Labs, Burlingame, CA) and diaminobenzidine (DAB) color detection systems (Vector Labs) according to the manufacturer's protocol. Sections were counterstained with Hematoxylin QS (Vector Labs), dehydrated, cleared in xylene, and mounted with DePeX (BDH; Laboratory Supplies, Melbourne, Australia). For PCNA detection, we used mouse anti-PCNA (Dako) at 1:1,000 dilution (without counterstain), and for cleaved caspase-3, we used rabbit polyclonal antibody (Cell Signaling, Beverly, MA) at 1:1,000 dilution (counterstain methyl green). All slides were examined using an Olympus BH2 microscope (Japan) for bright-field microscopy.
To assess the degree of muscularization of small pulmonary arterioles, lung slides of four randomly chosen animals in each treatment group were examined. In each slide, 10 high-power (x200) fields were selected at random at the level of distal acini, and vessels having an approximately circular profile with their external diameters less than 50 µm were identified. From these, the number of completely muscularized vessels were counted. Measurement of muscle thickness was assessed using a calibrated eye piece and assessing the mean smooth muscle thickness of 30- to 50-µm vessels (4 rats/group x 15 vessels/rat). For PCNA and cleaved caspase-3 detection, 5 high-power fields were examined, and 15 vessels less than 50 µm/animal were counted.
Statistical analysis. Data are expressed as means ± SE. Multiple comparisons between group means were performed using the nonparametric Kruskal-Wallis H test. When an H value indicated a significant difference, pairwise comparisons between group means were performed using the protected rank sum test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Effect on vascular muscularization.
To assess the effect of BMPR2 transgene expression on hypoxia-induced vascular remodeling, 10 high-power lung fields were examined in four rats from each treatment group in which the number of completely muscularized vessels less than 50 µm were counted. Chronic hypoxia almost tripled the number of muscularized vessels compared with normoxia (56 ± 4 vs. 21 ± 1, respectively, P < 0.05, Fig. 8A). A similar effect was seen on smooth muscle layer thickness (Fig. 8B). This effect was unchanged by control Ad vector delivery, whereas targeted BMPR2 gene delivery significantly attenuated the hypoxia-induced increase in vascular muscularization by 
40% (P < 0.05). Lung tissue sections, representative of each treatment group, immunostained for smooth muscle -actin, are shown in Fig. 9, A–D.
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DISCUSSION |
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The results of the current study are significant in a number of respects. The data presented helps to broaden our understanding of the impact of BMPR2 signaling in pulmonary hypertension beyond the relatively rare setting of inherited genetic mutations in BMPR2. Evidence that BMPR2 might be important beyond familial PAH was first noted by the study of Atkinson et al. (1), where reduced expression of BMPR2 in secondary as well as primary pulmonary hypertension was noted. Similarly, in the hypoxic rat model of PAH, chronic hypoxia has been shown to decrease BMPR2 expression in the pulmonary vasculature (26). More importantly, the current data show that upregulation of BMPR2 signaling can achieve a therapeutic outcome, which broadens the therapeutic possibilities for PAH therapy. Currently available treatments do not specifically address BMPR2 signaling; thus, this new approach could potentially be pursued to achieve either synergistic or additive advances on current practice. Although we have used a gene-based approach herein, the data also provide a rationale for the development of conventional pharmaceuticals that may achieve the same end. Nevertheless, the study is novel in its use of a pulmonary vascular-targeted, gene-based approach to treatment delivery, and gene therapy for patients with a recognized BMPR2 mutation would be an attractive proposition.
The precise mechanism by which BMPR2 mutations lead to PAH remains unclear. In general, the initial hypotheses were that BMPR2 activation is proapoptotic or antiproliferative, and this is consistent with the hypothesis that loss of activity in this pathway predisposes to abnormal proliferation as seen in PAH. The recent work by Yang et al. (35) indicates that the antiproliferative effect is mediated by SMAD signaling, at least in proximal pulmonary artery smooth muscle cells. This study also showed that there is a reduced proportion of cells expressing the activated form of SMAD1 (a downstream messenger for BMPR2 signaling) in the small pulmonary vessels of patients with both familial and idiopathic PAH (35). We have shown that our gene delivery approach does indeed upregulate SMAD transcriptional responses in vitro. However, in more distal pulmonary muscle cells, Yang et al. showed that BMP stimulation actually mediates smooth muscle cell proliferation, an effect mediated via p38 MAPK and ERK1/2 pathways. This finding is more difficult to reconcile with the pathological findings, and it is as yet unknown which pattern of response might best reflect the situation in the most distal small vessels, the primary sites of abnormality in PAH. Less is known about the role of BMPR2 expression in endothelial cells than smooth muscle cells in this process. Endothelial expression may be critical, particularly as in the human lung BMPR2 is predominantly expressed in the endothelium, with lesser amounts in the smooth muscle cells. Our current immunohistochemical studies of transduced rat lung and those previously published with reporter genes using the ACE-targeting approach (including electron microscopy) show that it is the pulmonary vascular endothelium that is predominantly being transduced (20). Recently, Teichert-Kuliszewska and colleagues (28) showed that BMP signaling via BMP2 and BMP7 actually protected endothelial cells from apoptosis induced by serum starvation in vitro. They also showed that downregulation of BMPR2 by small interfering RNA led to an increase in apoptosis. Thus, it appears that BMP signaling, in certain contexts, has differential effects on smooth muscle cells and endothelial cells, at least in vitro. From this, it has been proposed (but not shown) that BMPs may exert a prosurvival or antiapoptotic effect in the endothelium in vivo (25). If correct, loss of this effect could then lead to disordered vascular integrity and PAH development. In principle, support for this notion comes from studies of anti-VEGF treatment (which reduces endothelial survival): endothelial cell death is followed by proliferation of apoptosis-resistant endothelial clones with development of severe pulmonary hypertension in the rat hypoxia model (27). However, a stage of increased endothelial apoptosis has not been described in PAH patients. The effects of BMP signaling, like all TGF- family members, are highly context specific. In our studies, we have shown both in vitro and in vivo that BMPR2 transduction led to an increase in endothelial apoptosis. The mechanism by which this then leads to a reduction in the pulmonary hypertensive response is unclear and requires further study of endothelially derived mediators such as endothelin. We have found no evidence for an endothelial protective effect.
Recently, McMurtry et al. (11) investigated nebulized delivery of BMPR2 using an Ad vector in the monocrotaline model of pulmonary hypertension and found no effect on established pulmonary hypertension in this setting. Their study differs from ours in a number of important respects. The mechanism of PAH development in their model is quite different than that for the hypoxia model, and responses to therapies may vary. Opposite effects of TIMP-1 gene delivery in the hypoxia model vs. monocrotaline model have previously been reported (29, 30). The method of gene delivery is also quite different, with airway epithelial and vascular smooth muscle cells being the major transduced cells via the airway route in contrast to endothelial cells via our ACE-targeted approach. The McMurtry group focused on established disease, whereas we have first investigated mechanisms in a preventive context. All pulmonary hypertension models are a compromise, and the hypoxia model we have used in the current study has acknowledged limitations with regard to its correlation to human PAH. Specifically, the plexiform lesions characteristic of idiopathic and familial PAH are not seen. Nevertheless, hypoxic pulmonary vasoconstriction and vascular remodeling are contributing factors in a broad range of conditions leading to PAH in clinical settings. The failure of nebulized BMPR2 gene therapy in the monocotaline model does not exclude the possibility that endothelial-targeted BMPR2 therapy might have benefits in inflammatory conditions. The recent study by Song et al. (24) showing that BMPR2 heterozygous mice are more susceptible to inflammatory stimuli provides a basis to hypothesize that BMPR2 therapy may have utility in inflammation-induced PAH.
The clinical application of gene therapy has been a major challenge. Vector technology continues to be a limiting factor, and advances in vecterology per se are a major focus of gene therapy research at present. The therapeutic gains that can be achieved by specifically targeted vectors have recently been demonstrated in a systemic hypertension model where vascular targeted eNOS gene delivery achieved a therapeutic impact, but untargeted vector did not (12). We have recently also shown that therapeutic gains are achieved with pulmonary vascular targeting of eNOS gene delivery compared with untargeted vector in the rat hypoxia pulmonary hypertension model (19). In the present study, we thus chose to use our current state-of-the-art technology to examine the effects of BMPR2 gene delivery. In the context of established pulmonary hypertension, the ACE-targeting approach could prove particularly useful as ACE is known to be upregulated in areas of vascular remodeling (15). However, considerable further refinement of the vector approach would be required for possible use in human disease. One of the drawbacks of the current system is the "two-component" approach (Ad + antibody) that makes clinical grade formulation difficult. Hence, a number of groups are working toward the generation of genetically modified vectors whereby the targeting ligand is incorporated into the vector particle (14, 33). In our current system, we have used a first-generation Ad that is well known to have proinflammatory effects due to low-level expression of viral genes within transduced cells and the stimulation of T cell-mediated cytotoxicity. This problem often leads to a relatively short duration of transgene expression. To circumvent this problem, helper-dependent Ads have been developed that lack viral sequences in their genome (22). It has recently been shown that these agents achieve longer transgene expression with less inflammatory response after vascular gene delivery (31). In other model systems, more than 2 yr of expression after a single injection has already been demonstrated (22). Despite the foregoing concerns, we did not note significant inflammation in our model, consistent with our previously published studies using reporter genes (18, 20). Thus, even at its current stage of development, our vector approach provides a useful strategy for the initial evaluation of candidate therapeutic genes for pulmonary vascular disease. The approach could also be used to assess the impact of other TGF- family genes implicated in PAH, such as ALK1 (6).
In conclusion, the current work provides new insights into the pathogenesis of pulmonary hypertension, illustrates the usefulness of a novel platform technology for the evaluation of candidate therapeutic pathways, and specifically indicates the potential for BMPR2 modulation to be considered as a future therapeutic option for the development of new treatments for this devastating disease. With further vector refinement, a gene therapy approach is feasible.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 compared with respective No BMP4. RLU, relative light units.
P < 0.05 for hypoxia control Ad+Fab-9B9 vs. hypoxia AdCMVBMPR2myc+Fab-9B9.
