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J Am Coll Cardiol, 2000; 35:1040-1047 © 2000 by the American College of Cardiology Foundation |
a Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Manuscript received June 7, 1999; revised manuscript received October 15, 1999, accepted November 19, 1999.
Reprint requests and correspondence: Dr. Hiroaki Shimokawa, Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
shimo{at}cardiol.med.kyushu-u.ac.jp
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Abstract |
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This study was designed to examine whether or not adenovirus-mediated gene transfer of C-type natriuretic peptide (CNP) can prevent coronary restenotic changes after balloon injury in pigs in vivo.
BACKGROUND
Gene therapy to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) might be useful but requires a method applicable for in vivo gene delivery into the coronary artery as well as the efficient vector encoding a potent antiproliferative substance. We tested whether the adenovirus-mediated gene transfer of CNP by use of an infiltrator angioplasty balloon catheter (IABC) might prevent the coronary restenotic changes after balloon injury.
METHODS
Balloon angioplasty was performed in the left anterior descending and the left circumflex coronary artery in pigs. Immediately after the balloon injury, adenovirus solution encoding either CNP (AdCACNP) or ß-galactosidase (AdCALacZ) gene was injected with IABC into the balloon-injured coronary segments. Expression of CNP was assessed by immunohistochemical staining and cyclic guanosine 3',5'-monophosphate (cGMP) measurement. Coronary restenotic changes were evaluated by both angiographic and histological examinations.
RESULTS
CNP was highly expressed in the media and the adventitia of the coronary artery at the AdCACNP-transfected but not at the AdCALacZ-transfected segment. In the AdCALacZ-transfected segment, vascular cGMP levels tended to be reduced as compared with the untreated segment, whereas in the AdCACNP-transfected segment, vascular cGMP levels were restored. Angiographic coronary stenosis was significantly less at the AdCACNP-transfected than at the AdCALacZ-transfected segment. Histological examination revealed that this was achieved primarily by the marked inhibition of the geometric remodeling of the coronary artery by the CNP gene transfer.
CONCLUSIONS
Adenovirus-mediated CNP gene transfer with the IABC system may be a useful gene therapy to prevent restenosis after PTCA in vivo.
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We have previously demonstrated that adenovirus-mediated gene transfer of dominant-negative H-ras (9), p21 (cyclin-dependent kinase inhibitor) (10) or C-type natriuretic peptide (CNP) (11) into the rat carotid artery suppresses the neointimal formation after balloon injury. Among these candidates, CNP (14,15) seems to be promising because it is secreted by endothelial cells and modulates vascular remodeling by a local autocrine/paracrine mechanism (16,17). Indeed, the endothelium-derived peptide has multiple effects, including dilation of blood vessels (18,19) and inhibition of VSMC proliferation and migration (20,21) through cyclic guanosine 3',5'-monophosphate (cGMP) cascade. In human advanced atherosclerotic arteries, endothelial CNP production is decreased (22), whereas VSMC overexpress CNP-specific receptor, natriuretic peptide receptor-B (NPR-B) (23). PTCA causes endothelial injury resulting in the loss of endothelium-derived antiproliferative factors, including CNP and nitric oxide (NO), both of which regulate the intracellular cGMP levels in vascular walls (24,25). Therefore, gene therapy to overexpress CNP may be effective for the site-specific treatment against proliferative vascular diseases, including restenosis after PTCA.
For the gene delivery system to the coronary artery, the infiltrator angioplasty balloon catheter (IABC) has recently been developed (26,27). It can deliver fluid directly into the normal porcine coronary artery with >90% efficiency with minimal vascular damage (26,27), and can be applied for an in vivo gene delivery system into the coronary artery.
Thus, the purpose of the present study was to examine whether or not local adenovirus-mediated gene transfer of CNP with the IABC system is an effective gene therapy to prevent the restenotic changes of the coronary artery after balloon injury in pigs in vivo.
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Methods |
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Experimental protocol. Experimental protocol is shown in Figure 1. Fifteen animals (pigs) underwent coronary balloon injury for both the left anterior descending (LAD) and the left circumflex (LCX) coronary artery, followed by in vivo gene transfer (CNP for one balloon-injured site and ß-galactosidase for another site in a randomized manner). The expression of CNP was examined by both immunostaining for the protein (n = 3) and measurement of vascular cGMP levels (n = 6) one week after the in vivo gene transfer, when adenovirus-mediated gene expression peaked (9–11), whereas coronary angiography and histological examination were performed in the remaining six animals three weeks after the gene transfer.
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IABC system. IABC is an angioplasty balloon catheter with 21 small nipples in three lines located on the surface of the balloon connected to the drug delivery port (26,27). This catheter has three lumens: one central lumen for the angioplasty guide-wire, one for balloon inflation and another for drug delivery (26,27). The balloon is 1.5 cm in length and 3.5 mm in diameter, with the injection nipples 0.25 mm in height and 0.25 mm in diameter. The drug delivery port was filled with virus solution until droplets appeared through the needles before use.
Animal preparation. Domestic male pigs (two to three months old, and weighing 25 to 30 kg) (Nihon Crea Inc., Tokyo, Japan) were sedated with intramuscular ketamine hydrochloride (1.25 mg/kg) and were then anesthetized with intravenous sodium pentobarbital (20 mg/kg). The animals were then intubated and ventilated with room air, and oxygen was supplemented via a positive-pressure respirator (Shinano Inc., Tokyo, Japan). Under aseptic conditions, the left carotid artery was surgically exposed, and a 10F sheath was inserted. After systemic heparinization (10,000 U/body), a preshaped Judkins catheter (10F; Medtronic Inc., Minneapolis, MN) was inserted into the left carotid artery, and coronary angiography in a left oblique view was performed using the Toshiba cineangiography system (DG-15GB/ CAS-CA; Toshiba Medical Inc., Tokyo, Japan). Electrocardiograms in leads I, II, III, V1 and V6 were recorded (San-Ei Polygraph System; NEC, Tokyo, Japan), and the arterial pressure was continuously measured with a pressure transducer (Gould Inc., Cleveland, Ohio) connected to a Judkins catheter throughout the experiment. After the virus solution was injected into the coronary artery, angiography was repeated. Finally, the carotid artery was ligated, the skin closed and the animals allowed to recover from anesthesia.
Balloon injury followed by adenovirus-mediated in vivo gene transfer into the porcine coronary artery. The coronary artery was injured with a conventional balloon catheter with a diameter 1.3 to 1.5 times larger than coronary diameter by inflating it five times for 30 s at eight atmospheres in both LAD and LCX. We previously confirmed that the extent of coronary lesion induced by balloon injury is comparable between LAD and LCX in porcine coronary arteries (32). After this procedure, adenoviral gene transfer was performed at the previously injured coronary segment. IABC was advanced to the injured coronary artery followed by the inflation of a 3.5-mm balloon at two atmospheres. AdCALacZ (final titer, 4 x 108 pfu in 0.4 ml sorbitol-added lactated Ringer’s saline) and AdCACNP (final titer, 4 x 108 pfu in 0.4 ml sorbitol-added lactated Ringer’s saline) were randomly injected into the injured LAD and LCX. After the gene transfer, IABC was deflated and withdrawn, and the left carotid artery ligated.
Coronary angiography and coronary diameter measurement. Coronary angiography in the left anterior oblique view was performed before and three weeks after the gene transfer. Coronary stenosis of the balloon-injured segments was expressed as the percent decrease in the luminal diameter compared with the mean diameter of the adjacent proximal and distal normal coronary segments after the intracoronary administration of nitroglycerin (10 µg/kg).
Histochemical analysis for ß-galactosidase. Histochemical analysis for ß-galactosidase was performed one week after the injection of AdCALacZ into the coronary arterial wall. The animals were then killed with a lethal dose of sodium pentobarbital, exsanguinated and then the coronary artery was excised and fixed in phosphate-buffered saline (containing 2% formaldehyde and 0.2% glutar-aldehyde) for 2 h at 4°C. After the fixation, the coronary arteries were evaluated for LacZ expression by staining with a choromogenic substrate (X-gal; Wako Chemicals, Tokyo, Japan). For the light microscopic examination, tissue samples were embedded in paraffin after X-gal staining, sectioned into slices 5 mm thick, mounted on glass slides and double-stained with hematoxylin-eosin and nuclear fast red.
Histochemical analysis for CNP. Histochemical assays for CNP were performed one week after the injection of AdCACNP into the coronary artery. After the coronary artery was excised, it was quickly frozen in OCT compound, sectioned at 5 µm and subjected to immunohistostaining with polyclonal antibody against CNP (Peninsula Lab., Belmont, California). Intact arteries and nonimmune rabbit IgG were used as controls. Immunoreactive materials were visualized by use of a biotinylated anti-rabbit IgG antibody (Wako Chemicals, Tokyo, Japan), peroxidase-labeled streptavidin and diaminobenzidine.
Radioimmunoassay for cGMP. To examine the production of CNP, cGMP levels in a coronary artery were measured in frozen sections from AdCACNP- and AdCALacZ-infected injured coronary segments, as well as in those from uninjured normal coronary arteries. Vessels from the animals were removed one week after the gene transfer. All frozen tissues were homogenized in 0.1N hydrochloric acid, and centrifuged. The supernatants were assayed by radioimmunoassay.
Histological examination. Three weeks after the gene transfer, the heart was removed and the left coronary arteries were perfused with 6% formalin at the pressure of 120 mm Hg and fixed with formalin for one week. For the light microscopic examination, tissue samples were embedded in paraffin, sectioned into slices 5 µm thick, mounted on glass slides and stained with hematoxylin-eosin and van Gieson’s methods. With a photomicroscopic photograph system (Microphot-FXA; Nikon Co., Tokyo, Japan), pictures of coronary arteries were taken at 20x and 40x magnifications. In each specimen, lumen area and an area encircled by the internal elastic lamina (IEL) or the external elastic lamina (EEL) was measured with an automated computer-based image analyzer (Digitizer KD4600; Graphtec Corp., Yokohama, Japan) (32,33). The degree of intimal thickening was assessed by the ratio of intimal area to IEL area (% intima) (32,33). Coronary geometric (constrictive) remodeling was assessed by measuring the ratio of the EEL, IEL and lumen areas at the balloon-injured coronary segments to the mean of those of adjacent proximal and distal normal coronary segments (32,33). The extent of balloon injury was assessed by the ratio of the fractured IEL to the whole IEL length (32,33).
Data analysis. All results were measured by experienced observers blinded to the origin of the samples, and expressed as the mean ± SEM. Paired data were analyzed by paired t test, and multiple means were analyzed by one-way analysis of variance, followed by Fisher’s post hoc test. A p < 0.05 was considered to be statistically significant.
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Discussion |
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CNP as a promising candidate of gene therapy. Endogenous CNP is secreted from vascular endothelial cells, acting as an endothelium-derived relaxing and antiproliferative peptide (17,20,21). Unlike ANP and BNP, which regulate body fluids and blood pressure as cardiac hormones (34), CNP is involved in the vascular natriuretic peptide system, modulating the phenotype of VSMC, and thus regulating vascular remodeling through activation of cGMP cascade (17,20,21). In the present study, the expression of CNP was highly detectable by the immunohistochemical staining in the media and the adventitia of the porcine coronary artery after the transfection with AdCACNP. It is considered that the production of CNP from VSMC, fibroblast or other inflammatory cells resulted in the increase in vascular cGMP levels in the coronary artery. Although other CNP-mediated mechanisms might also be involved in the inhibition of vascular remodeling in this study, it is highly possible that CNP/cGMP cascade primarily regulated the vascular remodeling by inhibiting the proliferation and migration of VSMC.
The advantages of CNP as a candidate for gene therapy include its local but diffusable effect. Indeed, in the present study, the biological effects of CNP were localized in the site at which AdCACNP was transfected without causing any systemic effect such as hypotension or natriuresis (data not shown). The short half-life of CNP in the plasma (35) and the overexpression of CNP receptor (NPR-B) in injured artery (22,23) may explain the site-specific effects of CNP. Unlike the expression of control gene (AdCALacZ), the immunoreactivity of CNP was noted throughout the media and the adventitia, indicating that CNP, once produced and secreted by infected cells (e.g., VSMC and fibroblasts), diffused throughout the vessel wall and exerted its antiproliferative effect throughout the vessel wall. NO may share the same characteristics as CNP. Indeed, Varenne et al. (36) recently reported that local adenovirus-mediated transfer of eNOS reduced luminal narrowing after coronary angioplasty in pigs. However, overexpression of eNOS may have some limitations because it has been recently demonstrated that serious vascular smooth muscle tolerance to NO occurs in transgenic mice overexpressing eNOS (37).
Vascular remodeling vs. neointimal formation. It is now widely accepted that luminal narrowing after coronary angioplasty is caused primarily by geometric (constrictive) remodeling but not by neointimal formation (2–5). We have previously demonstrated that adventitial inflammatory/proliferative responses play an important role in the pathogenesis of the geometric remodeling in pigs in vivo (32,38). Indeed, when tyrosine kinases, which are the key step for proliferative responses, were inhibited by the adventitial treatment with a specific inhibitor, the development of geometric remodeling caused by either balloon injury (32) or platelet-derived growth factor (38) was markedly inhibited. Thus, the adventitial delivery of an antiproliferative gene with IABC in the present study appears to be a reasonable approach to prevent the geometric remodeling. By contrast, the neointimal formation was mild and apparently did not significantly contribute to the luminal narrowing in the present model of coronary angioplasty. However, it remains to be examined why the neointimal formation was not significantly inhibited at the AdCACNP-transfected site.
IABC as a gene delivery system into the coronary artery in vivo. The volume of adenovirus vector used in the present study with IABC (4 x 108 pfu in 0.4 ml) was one-tenth of that used in the previous studies with a percutaneous balloon catheter (39,40), demonstrating a high efficiency of the IABC system for in vivo gene transfer into the coronary artery. Indeed, it is important for this type of gene therapy to use a small amount of vector solution to avoid the local inflammation and systemic responses (26,27). A more sophisticated catheter needs to be developed for more efficient gene delivery into the coronary artery in vivo.
Limitations of the present study. Several limitations could be raised for the present study. First, the present study was performed in the otherwise normal porcine coronary artery. In the atherosclerotic coronary artery with an increased wall thickness, the present approach with IABC may have a limitation; however, the expression of diffusable antiproliferative factor (e.g., CNP, NO) with a more sophisticated infiltrator catheter may overcome this problem in the future. Second, the use of adenovirus vector induces an intrinsic immunity against the vector, and therefore repeated use of the present strategy may be limited. Thus, the inhibition of such an immunity by a suitable method (e.g., temporal use of immunosuppressant) and the development of a less immunogenic vector remain to be examined (41). Third, the use of IABC itself may cause a greater vascular injury than conventional balloon catheters. However, the extent of vascular injury (fractured IEL) was comparable between the present study with IABC and the previous study with a conventional balloon catheter (32). Indeed, IABC is now widely used for coronary intervention in European countries without any major complications (27). Fourth, the extent of neointimal formation was too mild to examine the inhibitory effect of CNP. The greater extent of balloon injury would also reveal the inhibitory effect of CNP gene transfer on the neointimal formation.
In summary, the present study demonstrated that the local adenovirus-mediated transfer of CNP suppresses vascular remodeling in porcine coronary arteries in vivo. This strategy might also be useful to prevent restenosis after PTCA in humans.
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