Published ahead of print on March 16, 2006, doi:10.1165/rcmb.2005-0115OC
© 2006 American Thoracic Society DOI: 10.1165/rcmb.2005-0115OC Microvascular Regeneration in Established Pulmonary Hypertension by Angiogenic Gene TransferThe Terrence Donnelly Vascular Biology Laboratories, Division of Cardiology, St. Michael's Hospital, and Department of Medicine and the McLaughlin Center for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada Correspondence and requests for reprints should be addressed to Dr. Duncan J. Stewart, Dexter Hung-Cho Man Chair and Director of the Division of Cardiology, University of Toronto, St. Michael's Hospital, 30 Bond Street, Suite 6-050k Queen Wing, Toronto, ON, M5B 1W8 Canada. E-mail: stewartd{at}smh.toronto.on.ca
Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 106 syngeneic fibroblasts (FB) transfected with vascular endothelial growth factor A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day 21, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles 21 d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.
Key Words: cell-based gene transfer • eNOS • gene therapy • pulmonary hypertension • monocrotaline • vascular regeneration • vascular endothelial growth factor
Pulmonary arterial hypertension (PAH) is caused by progressive increases in pulmonary vasculature resistance leading to elevations in pulmonary arterial pressure. Idiopathic (I) PAH, a rare form of this disease with unknown etiology, has a particularly poor prognosis with a mean life expectancy of less than three years after diagnosis (1). Although arterial vasoconstriction may play a role in some patients with PAH, the vast majority of patients with IPAH show little or no acute response to vasodilators, consistent with a dominant role of pulmonary arteriolar remodelling in the later stages of this disease (1, 2). Present therapies for IPAH have only a modest impact on pulmonary hemodynamics (3) and there is no evidence that they result in significant improvement in the pathologic changes in the pulmonary arterial bed. Unfortunately, most patients are not diagnosed until late in the course of the disease, at which time advanced arterial narrowing and loss of the pulmonary microcirculation predominates (1). Thus, new therapeutic approaches capable of reversing vascular structural changes and regenerating pulmonary microvasculature are needed to restore pulmonary hemodynamics in advanced PAH. Whether vascular regeneration is indeed possible in the pulmonary circulation is controversial. Although there is extensive literature demonstrating a role for angiogenesis in systemic vascular beds, this has not been well recognized in the pulmonary circulation. When lung angiogenesis occurs in the context of tumors or abscesses, it is thought to originate solely from the bronchial circulation (4, 5). Recently, gene transfer of angiogenic factors has been shown to be effective in preventing PAH in experimental models (6–8), whereas the inhibition of vascular growth factor receptors has been reported to potentiate both PAH and pathologic vascular remodeling in response to chronic hypoxia (9). These observations have led to suggestions that angiogenic factors may play a "protective role" in PAH (6–8), but the precise mechanism of benefit remains to be fully defined. In addition to the classical angiogenic factors, such as vascular endothelial growth factor (VEGF), there is increasing evidence implicating nitric oxide (NO) as a critical mediator of angiogenesis both in vitro and in vivo (10–14). NO is a potent stimulator of endothelial proliferation and migration, and endothelial NO synthase (eNOS) has been implicated as a downstream mediator of VEGF in the angiogenic cascade. Therefore, the aim of the present study was to define the efficacy of angiogenic gene therapy with VEGF and eNOS in established PAH. We used a "reversal" model in which gene therapy was delayed until 3 wk after monocrotaline (MCT) administration, at which time PAH and the associated vascular remodelling was already well developed (5, 15). In addition, we employed a novel microangiographic method to visualize the three-dimensional architecture of the pulmonary microcirculation to better assess the contribution of microvascular regeneration to any improvement in pulmonary hemodynamics in the different treatment groups. We now report that although both eNOS and VEGF gene transfer reduced the progression of PAH, only eNOS significantly reversed established PAH in the MCT model, at least in part by inducing the regeneration of the pulmonary microcirculation.
Cell Isolation and Culture Fisher 344 rats (Charles River Co., St. Constant, PQ, Canada) underwent skin biopsy, and fibroblasts (FBs) were cultured using an explant technique (8). Cells were grown in Dulbecco's Modified Eagle Media (DMEM) with 10% FCS and 2% penicillin/streptomycin (5,000 U penicillin G sodium/ml; 5,000 µg streptomycin sulfate/ml) in a humidified incubator (20% O2, 5% CO2 at 37°C).
Fluorescent Cell Labeling
Transfection
Animal Model Twenty-one days after MCT injection, the rats were anesthetized, and a 3F Millar microtip catheter was inserted via the right external jugular vein and into the right ventricle to obtain baseline measurements of right ventricular systolic pressure (RVSP) (Biopac System, Goleta, CA). Via the contralateral indwelling catheter, rats received three sequential injections of 5 x 105 transfected FBs at 24-h intervals (total dose = 1.5 x 106 cells). The indwelling catheter was then removed, the left external jugular vein was ligated, and animals were allowed to recover. Fourteen days after the injection of transfected FBs, animals were re-anesthetized. A 3F Millar microtip catheter was re-inserted to record RVSPs. Animals were killed and the left lung inflated with OCT and cut into sections for fixation (4% paraformaldehyde–0.1% glutaraldehyde PBS solution) and for paraffin embedding, or frozen for cryostat sectioning. The right lung was snap-frozen in liquid nitrogen for RNA extraction. The heart was microdissected for the assessment of right to left ventricular plus septal weight ratio (RV/LV).
RT-PCR
Quantitative PCR. Real-time PCR (ABI 7900HT; Applied Biosystems, Foster City, CA) was performed for human eNOS transgene expression, as well as for the endogenous expression of eNOS, VEGF, Flk-2 (VEGFR2), and angiopoietin-1 (Ang1), using 2x Taq Universal Master Mix with a dual-labeled probe: 6-FAM (6-carboxyfluorescein) and 6-TAMRA (6-carboxymethylrhodamine; both from PCR Core Reagents, Applied Biosystems). Quantification of the transcript was determined using a standard curve constructed from cDNA produced by a two-step conventional PCR reaction comprising an initial denaturation step at 95°C for 5 min and 18 cycles at 95°C (30 s), 50°C (45 s), and 72°C (45 s), followed by a final cycle at 72°C for 2 min. DNA content was determined by optical density and copy number was calculated using the molecular weight of the PCR product. Quantitative RT-PCR was performed in a 30-µl reaction using 2 µl of complementary cDNA from the original RT reaction. The standard curve and target genes were amplified using the primers shown in Table 2. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 40 cycles at 95°C for 15 s and 62°C for 1 min. All measurements were performed in duplicate.
Fluorescent Microangiography At 21 or 35 d after MCT, the lungs from an additional 27 rats were prepared for fluorescent microangiography (FMA). A catheter was inserted into the pulmonary artery and the lungs flushed with heparinized PBS at 37°C, immediately followed by perfusion with a warmed (45°C) solution of 1% low-melting-point agarose (Sigma-Aldrich, Oakville, ON, Canada) containing 0.2 µm yellow-green fluorescent microspheres (505 nm/515nm peak excitation and emission; Molecular Probes) at a concentration of 0.2% total solids. The agarose solution was injected until agarose could be seen flowing out of an incision in the left atrial appendage. The lungs were then fixed in 4% paraformaldehyde in PBS for 48 h at 4°C, and 150- to 200-µm-thick sections were prepared on an oscillating blade microtome (Leica, Wetzlar, Germany). Some sections were also counterstained with the nuclear marker propidium iodide (Sigma-Aldrich) to identify viable tissue structure. Confocal optical sectioning (BioRad Radiance, Hercules, CA) was used to produce a Z stack of images spanning 150 µm of section depth, and projections of Z-series images were created to demonstrate vessel architecture throughout the entire thickness of the section. A separate series of experiments were performed to assess the potential acute vasodilator effects of NO in this model. Twenty-one days after MCT, rats were treated with nitroglycerine (NTG 10 mg/kg, n = 6) administered by intraperitoneal injection 15 min before killing. Lungs were then perfused with fluorescent agarose suspension as described above. Another control group (n = 5) was treated in an identical fashion, but in the absence of NTG administration.
Quantitation of Fluorescence Intensity
Histology/Immunohistochemistry
Statistical Analysis
Engraftment and Persistence of Transplanted FBs Multiple CMTMR-positive cells were seen within the pulmonary circulation 15 min after jugular injection (Figure 1A, panels a and d), mainly in the lumen of small pulmonary arterioles and capillaries. By 1 and 5 d after injection, transplanted cells were seen mainly adjacent to small vascular structures (Figure 1A, panels b, c, e, and f). No positive cells were seen in any of the lungs injected with nonlabeled FBs. Quantification of injected fluorescently labeled FBs showed that by 24 h the number of fluorescently labeled cells was reduced to 59% of that seen at 15 min (Figure 1B), and by 15 d this was reduced to  25%, with little further decline seen by 30 d.
Time-Course of Transgene Human eNOS and VEGF Expression Using primers designed to selectively amplify plasmid-derived transcripts, peNOS or pVEGF was detected in all animals 14 d after receiving transfected cells (Figure 2A), confirming persistent transgene expression in both groups. The expression of plasmid-derived eNOS RT transcript was further quantified by "real-time" PCR, and remained remarkably constant over the first 30 d after cell-based gene transfer, then gradually decreased but was still detectible 3 mo after injection (Figure 2B, n = 3/each group).
Effect of Gene Transfer on Hemodynamic Parameters In normal rats receiving only sham saline injections (i.e., no MCT or transfected cells), RVSP was 28.4 ± 0.8 mm Hg on Day 21, and 28 ± 0.19 mm Hg at Day 35. In rats treated with MCT, PAH was well established by Day 21 after MCT, with the mean RVSP of 49 ± 1.5 mm Hg (P = 0.001 versus Day 21 normal) (Figure 3A). By Day 35, the group receiving null-transfected cells demonstrated a further increase in RVSP from 47 ± 3.5 to 64.5 ± 2.8 mm Hg (P = 0.0007). However, in the eNOS-treated group, RVSP on Day 35 (40.7 ± 1.6 mm Hg) was significantly lower in the same animals than on Day 21 (50.2 ± 1.9 mm Hg, P = 0.0001). Although VEGF gene transfer reduced the progression of PAH, there was no significant difference in RVSP between Day 35 (50.3 ± 2.4 mm Hg) and Day 21 (50 ± 3.0 mm Hg; P > 0.05) in the VEGF-treated group.
The ratio of the right ventricular to left ventricular + septal weight (RV/LV) was used as a measure of right ventricular hypertrophy (Figure 3B). MCT treatment resulted in severe right ventricular hypertrophy, with a significant increase in RV/LV (0.43 ± 0.06) in the null transfected group compared with 0.25 ± 0.02 in the normal controls (P < 0.001 versus normal). RV/LV was reduced in both the peNOS- and pVEGF-treated groups (0.31 ± 0.01 and 0.28 ± 0.01, respectively) compared with null-transfected animals (P < 0.05).
FMA of the Pulmonary Vasculature
Pulmonary microvascular PI was significantly reduced in the MCT-treated lungs (Figure 5A) compared with the normal controls. Animals receiving eNOS gene transfer showed a marked improvement in capillary perfusion (P < 0.001 versus MCT), whereas the lungs of VEGF-treated rats showed an intermediate pattern, with significant residual regions of arteriolar–capillary discontinuity (ns versus MCT). A similar pattern was observed when the ratio of arterioles per 100 alveoli was determined for the same treatment groups (Figure 5B).
Arteriolar Muscularization and Remodeling MCT-treated rats exhibited medial hypertrophy of small arteries that could easily be appreciated on light microscopic examination of hematoxylin and eosin–stained lung sections (Figure 6A, panels a and b). The proportion of muscularized arterioles was determined using dual fluorescent immunostaining for vWF and  smooth muscle actin (Figure 6A, panels c and d). In normal lungs, arterioles of < 30 µm showed infrequent muscularization, with only 26.3 ± 8.2% demonstrating PM and 6.5 ± 3.2% with FM. In contrast, MCT-treated animals receiving the null vector showed a substantially greater proportion of small arterioles with PM or FM (P < 0.01 versus normal controls, Figure 6B). Gene transfer with eNOS reduced the percentage of small arterioles exhibiting muscularization (P < 0.01 versus MCT-null), whereas MCT rats treated with VEGF-transfected cells demonstrated no significant decrease in the muscularization of small vessels.
Angiogenic Gene Expression Administration of MCT alone produced a profound decrease in the endogenous expression of eNOS and a variety of angiogenic genes including VEGF, Flk-1, and Ang1 (Figure 7). Cell-based gene transfer of eNOS partially restored the expression of these angiogenic genes, whereas VEGF had no significant effect.
The present report demonstrates that cell-based gene transfer with eNOS produced a significant improvement in established PAH in the MCT model, while restoring a more normal pattern of pulmonary microvasculature and alveolar capillary perfusion. These findings support reestablishment of pulmonary arteriolar–capillary continuity as an important mechanism for improvement of the hemodynamic abnormalities in experimental PAH, and suggest that the regeneration of pulmonary microcirculation could represent a novel treatment paradigm for this progressive and lethal disorder. The observation that angiogenic gene therapy was associated with the improvement in microvascular architecture in the MCT model has potentially important implications for the therapy of PAH since, in its advanced stages, this disease is characterized by widespread arteriolar pruning and loss of pulmonary microcirculation (1, 17). While angiogenesis in systemic vascular beds has been well documented in models of chronic ischemia (13), there have been no prior reports demonstrating vascular regeneration in reponse to angiogenic factors in the pulmonary circulation. In conditions in which angiogenesis does occurs in the lung, such as in neoplasia, new vessels are thought to arise from the bronchial rather than the pulmonary arterial tree (4, 5, 18). However, in the present study, microangiography of the pulmonary circulation clearly demonstrated the restoration of continuity between the distal pulmonary arterioles and the alveolar capillary bed in response to eNOS gene transfer in MCT-treated rats. At the very least this finding is consistent with the "recanalization" of pre-capillary arterioles, which likely involves migration and proliferation of the adjacent endothelial cells in reponse to stimulation with NO and VEGF. The present report adds to the recent findings that transplantation of endothelial progenitor cells (EPCs) inhibits the progression of MCT-induced PAH in both the prevention (19, 20) and reversal models (20). EPCs are released from the bone marrow into the circulation, homing to regions of ischemia or vascular damage, and are thought to participate directly and indirectly in angiogenesis and vascular repair (21). Thus, the ability of EPCs to prevent PAH in this model provides further evidence supporting angiogenesis as a potentially therapeutic mechanism for PAH. Of note, EPCs alone did not produce significant improvement in established PAH (20), and only eNOS-transfected EPCs were effective in reducing pulmonary arterial pressure and re-establishing capillary perfusion in the reversal model (20). However, it is quite likely that eNOS enhanced the regenerative potential of these progenitor cells, possibly by upregulating the expression of angiogenic cytokines, and thus no firm conclusions can be drawn about any direct beneficial effects of eNOS gene transfer in extablished PAH from this report. In contrast, in the present study, the therapeutic benefit can be ascribed entirely to gene therapy, since transplantation of somatic fibroblasts resulted in no improvement by itself. Recently, bone marrow–derived mononuclear cells have been suggested to contribute to adverse adventitial (22) and medial remodeling (23), and thus it is possible that angiogenic gene therapy may have advantages over the use of stem and progenitor cell therapy. Although both eNOS and VEGF gene transfer reduced RVSP and RV remodeling at end-study compared with MCT alone, only eNOS resulted in a significant improvement in pulmonary hemodynamics in the same animals at Day 35 compared with Day 21. This greater efficacy of eNOS gene therapy compared with VEGF in reversing established PAH is intriguing and unexpected. In the prevention model, in which cell-based gene transfer was delivered at the same time as the administration of MCT, both transgenes produced very similar and near complete prevention of pulmonary hypertension and remodeling changes of the right ventricle and pulmonary arteries (6, 16). Although we cannot exclude the possibility that the apparent difference in the reversal model may merely reflect gene dosage or a difference in potency between the two strategies, similar levels of mRNA expression were documented for both transgenes 2 wk after cell therapy. It is also possible that because of its broader range of potentially beneficial actions, the therapeutic effects of NO may extend beyond those of VEGF (24). For example, NO has been shown both to inhibit the growth (25), and promote apoptosis (26), of vascular smooth muscle cell growth (VSCM), as well as inhibit platelet thrombosis (27). Although these actions may contribute to the overall hemodynamic improvement seen in the MCT reversal model, they do not explain the increase in vascularity at the pre-capillary and capillary levels seen in eNOS-treated animals compared with null-transfected rats in the MCT reversal model. It is being increasingly recognized that NO plays an important role in downstream signaling in response to a variety of angiogenic growth factors (10, 14), including VEGF (11, 28, 29). In addition, NO has been implicated in the regulation of angiogenic gene expression (30). Indeed, eNOS gene transfer improved the endogenous expression of VEGF and other angiogenic genes to a greater extent than VEGF. This is unlikely to have been due solely to an effect of eNOS on the transfected FBs themsleves, since the mass of transplanted cells could only account for a small proportion of the overall lung transcript level. Thus, it is likely that the increase in mRNA levels of these genes, including eNOS itself, reflected an improvement in the expression profile of the lung vasculature due to paracrine effects of the transfected cells or indirectly as a result of restoration of more normal vascular structure and function. Of particular relevance, our group has recently reported that eNOS may play a critical and previously unrecognized role in pulmonary vascular development during lung morphogenesis (31). The lung circulation is unique in that the arterial and capillary beds develop independently (32). Whereas the alveolar capillaries are thought to originate de novo by vasculogenesis, the arterial tree is believed to develop through sprouting angiogenesis and it is only in mid- to late gestation that these two beds fuse at the level of the pre-capillary arteriole (32). Of note, the lung phenotype of eNOS-deficient pups, which exhibit a high rate of neonatal mortality due to respiratory distress (31), was highly reminiscent of alveolar capillary dysplasia (ACD) (33–35), a clinical syndrome characterized by failure of arteriolar in-growth into the alveolar septae and misalignment of pulmonary veins (34, 35). Moreover, fluorescent microangiography of lungs from preterm fetal eNOS–/– pups revealed abnormalities in microvascular structure very similar to those of MCT-induced PAH. In particular, the demonstration of discontinuity of the distal arteriolar circulation is consistent with failure of normal fusion of the alveolar capillary bed. This unique developmental mechanism of the pulmonary circulation may also have important implications for postnatal pulmonary vascular diseases. The distal "pre-capillary" arterioles, which are the last to form during embryonic development, may also be particularly vulnerable to injury and degeneration in disease. Normally, these vessels lack medial smooth muscle cells, which are known to stabilize and protect systemic arterioles from regression (36), and exhibit only scant extracellular matrix. Thus, this critical vascular region may be uniquely susceptible to environmental stress, predisposing to microvascular degeneration, possibly as a consequence of endothelial apoptosis (9). This is also consistent with the characteristic location of arteriolar occlusions at the pre-capillary level as visualized by microangiography in experimental PAH in the current study as well as in human lung tissue from PPH lungs (17). The critical role of eNOS in lung vascular development may also partly explain the remarkable efficacy of eNOS gene transfer in reversal model of PAH compared with VEGF, since the same mechanisms by which this gene directs the fusion of the arteriolar and capillary beds during ontogeny (32) may also contribute the regeneration of this crucial junction in lung vascular disease. Thus, in the present study gene therapy with eNOS resulted in the reversal of established experimental PAH, which was associated with regeneration of pre-capillary arteriolar continuity and reestablishment of the alveolar capillary perfusion. These observations have important implications both for the pathogenesis and treatment of pulmonary vascular disease, and suggest that angiogenic gene transfer may represent an effective therapeutic paradigm for advanced disease.
* Present affiliation: Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois.  Originally Published in Press as DOI: 10.1165/rcmb.2005-0115OC on March 16, 2006 Conflict of Interest Statement: Y.D.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.W.C. serves as a consultant to Northern Therapeutics, a start-up biotechnology company involved in the development of cell based therapies for pulmonary disease. D.S.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.J.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.P.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.N.N.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.J.S. is the founding scientist of Northern Therapeutics, which was incorporated as a Canadian-controlled private company in January 2000. He acts as the Chief Scientific Officer, and for this he receives a stipend of $25,000 per year. He has a minority equity position in the company and as a Director receives share options of undetermined value. Received in original form March 23, 2005 Accepted in final form January 20, 2006
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Abstract
Introduction
P < 0.01 versus pcDNA. (B) The effect of cell-based gene therapy on the ratio of right ventricular to left ventricular plus septal weight (RV/LV) at end-study (Day 35). Rats receiving the null (pcDNA) vector showed a significant increase in RV/LV ratio at Day 35 after MCT injection compared with normal controls, indicative of right ventricular hypertrophy. RV/LV was similarly reduced by eNOS and VEGF gene transfer. 
