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Ann Thorac Surg 1996;62:425-433
© 1996 The Society of Thoracic Surgeons
Department of Cardiothoracic Surgery, Division of Pulmonary and Critical Care Medicine, and Division of Cardiology, The New York Hospital-Cornell Medical Center, New York, New York
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Abstract |
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Methods. Mongrel dogs underwent left thoracotomy and direct myocardial injections (100 µL/injection) of adenovirus vectors (109 pfu) carrying the DNA for the reporter enzyme chloramphenicol acetyl transferase or the angiogenic protein vascular endothelial growth factor. Two to 14 days after vector administration, regional protein expression was evaluated in myocardium and distant organs. Left ventricular function, assessed by echocardiography, and routine hematologic and biochemical indices were evaluated before and after vector administration.
Results. Peak levels of chloramphenicol acetyl transferase activity were detected 2 days after vector administration, and levels above baseline persisted for at least 14 days. Local chloramphenicol acetyl transferase activity was detected at distances at least as far as 1.5 cm from the site of injection. Chloramphenicol acetyl transferase activity in distant organs was less than 0.1% of that in injected myocardium 7 days after vector administration. Localized expression of vascular endothelial growth factor was achieved for up to 7 days after a single vector administration. Cardiac function and laboratory values were unchanged during the study.
Conclusions. Adenovirus-mediated direct myocardial gene transfer can be accomplished safely in a large animal model, providing high levels of protein expression in a greater spatial distribution than previously reported, with minimal transfection of distant organs. Sustained and localized expression of a potent angiogenic mediator has been accomplished, which may provide an innovative strategy to stimulate angiogenesis in ischemic myocardium.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Gene therapy, a modality involving the introduction of exogenous genes into living cells, represents an innovative method potentially capable of treating both inherited and acquired diseases of the cardiovascular system [1]. In vivo myocardial gene transfer strategies have included the administration of plasmid DNA and viral vectors by either coronary catheter infusion [2, 3] or direct myocardial injection [4, 5]. Experience to date with the direct delivery of viral vectors to myocardium is limited, however, and has been complicated by a narrow spatial distribution of gene expression [4]. Furthermore, most experience with this gene transfer strategy has been confined to the delivery of reporter genes.
A number of growth factors are known to elicit angiogenic responses in vivo [6], and the administration of these mediators in protein form to the myocardium has been shown to promote revascularization and salvage of ischemic tissue [7, 8]. Vascular endothelial growth factor (VEGF), a homodimeric 34- to 42-kDa heparin-binding glycoprotein, is a potent stimulator of endothelial cell replication and is regarded as one of the most specific of the known angiogenic mediators because the localization of its receptors is specific to endothelial cells [9]. Consistent with the hypothesis that VEGF plays a major role in the biologic process of angiogenesis are the observations that VEGF and VEGF receptor expression are upregulated in areas of myocardial tissue ischemia [10, 11]. Furthermore, local administration of recombinant VEGF protein has been shown to augment the development of collateral blood vessels and to improve cardiac function in experimental animal models of myocardial ischemia [12, 13].
In theory, successful use of VEGF or other angiogenic mediators would involve administration of the growth factor to a specific territory in vivo over a period of time sufficient to stimulate angiogenesis. Ideally, VEGF should remain localized to avoid inducing promiscuous angiogenesis in sensitive, nondiseased organs such as the retina or synovium, or in occult tumors. To accomplish this aim, the administration of VEGF continuously through indwelling catheters has been described in animal models of myocardial ischemia [12, 13]. These internal delivery systems, however, involve constant infusion over a number of days and might prove to be cumbersome in a clinical setting. In vivo gene transfer, using replication-deficient adenovirus vectors, has emerged as a safe and effective method of achieving targeted protein expression in a number of experimental models. More specifically, adenovirus vectors have been shown to be effective at transferring genes to myocardium with high levels of expression for at least 1 week [4, 14–16]. A single in vivo administration of the gene coding for VEGF or other growth factors packaged in an adenovirus vector may, therefore, provide an ideal strategy for achieving the goal of sustained, regional expression of a therapeutic angiogenic protein in a relatively inaccessible target organ such as the myocardium.
An angiogenic response after in vivo administration of an adenovirus vector containing the VEGF gene has been described recently in the subcutaneous tissue of mice [17]. To devise a gene therapy strategy using adenovirus vectors to induce myocardial angiogenesis, we sought first to define the pharmacokinetics of direct adenovirus-mediated myocardial gene transfer in large animals. In this regard, the present study was designed to do the following: (1) characterize the temporal and spatial limits of gene expression, (2) determine the safety of this gene transfer strategy, and (3) demonstrate the potential for sustained myocardial expression of a biologically relevant and potentially therapeutic protein such as VEGF.
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Material and Methods |
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). AdCMV.Null (similar to AdCMV.VEGF, but with no gene in the expression cassette) was used as a control vector for in vitro experiments [18]. AdCMV.CAT (similar to AdCMV.VEGF, but coding for chloramphenicol acetyl transferase) was used to transfer and express a marker gene [16]. All adenovirus vectors were propagated in 293 cells (American Type Culture Collection, Rockville, MD), purified by CsCl density purification, dialyzed, and titered by plaque assay, as described previously [19]. Vectors were stored in aliquots (50 µL) at -70°C.
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For coronary artery injections, a femoral arteriotomy was performed under sterile conditions and a 7.5F sheath was inserted. Under fluoroscopic guidance, an AR II 7F 100-cm catheter (Bard, Tewksbury, MA) was advanced to the left coronary ostium and positioned in the proximal left anterior descending artery, which was confirmed by the administration of radiopaque contrast. The adenovirus vector was administered in a volume of 1 mL over 10 seconds and flushed with 3 mL 0.9% NaCl. After vector delivery, the catheter position was again confirmed by administration of contrast, the catheter and sheath were removed, and the animals were permitted to recover.
Transfer of a Reporter Gene In Vivo
To confirm that adenovirus vectors are capable of transferring complementary DNA to myocardium in vivo and to determine an optimum technique of delivery, we administered the vector AdCMV.CAT (109 pfu) to canine myocardium by both direct injection (3 animals) and coronary catheterization (3 animals). Seven days after vector administration, the myocardium was evaluated for evidence of gene expression. Animals underwent a left thoracotomy as described earlier; the hearts were arrested in diastole with KCl (20 mEq) and excised. Coronary ostia were cannulated and flushed with phosphate-buffered saline solution. For each animal that had received an intracoronary vector administration, myocardium in the distribution of the left anterior descending artery was divided equally into 20 samples (0.5 to 1.0 g). Each of the 20 full-thickness samples per animal was then divided further into equal epicardial and endocardial halves, rinsed in phosphate-buffered saline solution, weighed, and stored at -70°C for subsequent evaluation of chloramphenicol acetyl transferase (CAT) activity.
For animals that had been administered vector by direct injection, the myocardium was divided into four equal full-thickness samples centered on the marking suture. Using a standard grid pattern, we further divided the samples into central (0 to 10 mm from the site of injection) and peripheral (11 to 15 mm from the site of injection) regions, and further divided these into epicardial and endocardial halves. The result was a total of 16 similarly sized samples of myocardium (0.5 to 1.5 g) per animal at representative distances from the site of vector administration. Based on this standard dissection technique, each sample was designated to be from one of the following four regions: (1) central epicardium (0 to 10 mm from the site of vector administration and epicardial half of the myocardium), (2) peripheral epicardium (11 to 15 mm from the site of vector administration and epicardial half of the myocardium), (3) central endocardium (0 to 10 mm from the site of administration and endocardial half of the myocardium), and (4) peripheral endocardium (11 to 15 mm from the site of administration and endocardial half of the myocardium).
To characterize the temporal and spatial limits of adenovirus vector-mediated gene expression in myocardium over time, we administered AdCMV.CAT (109 pfu) by direct myocardial injection. Tissue was harvested and regionally categorized, as described previously, immediately and 2, 5, 7, and 14 days after vector administration (3 animals per time point). Tissue samples were then evaluated individually for CAT activity.
To evaluate a dose response after direct myocardial administration of adenovirus vectors, we administered AdCMV.CAT in doses of 107, 108, and 109 pfu, each at three separate locations along the left ventricular free wall, as described earlier (3 animals). Seven days after vector administration, epicardial tissue samples (four per dose) were harvested from a distance of 1 to 10 mm from the site of injection, and CAT activity was quantified. In a separate naive (untreated) animal, four epicardial tissue samples were harvested and similarly quantified for CAT activity.
To define the extent of systemic adenovirus-mediated transfection 7 days after intramyocardial AdCMV.CAT vector administration (109 pfu; 3 animals), samples (0.5 to 1.5 g) of distant organs, including left lung, right lung, thymus, diaphragm, liver, spleen, kidney, gonad, skeletal muscle, and right ventricular myocardium, were obtained at the time of sacrifice and evaluated for CAT activity.
In each tissue sample, CAT activity was quantified by thin-layer chromatography and phosphorimager analysis [16]. When CAT activity was greater than 70%, samples were diluted in phosphate-buffered saline and reassessed until CAT activity was within the linear range. Chloramphenicol acetyl transferase activity was calculated from the percentage of acetylated chloramphenicol relative to the wet weight of the tissue, multiplied by the dilution factor of the cell lysate used to maintain the assays within the linear range, and reported as units per mg tissue.
Transfer of Vascular Endothelial Growth Factor cDNA In Vivo
To evaluate the feasibility of achieving sustained local levels of a therapeutic angiogenic protein in myocardium, we administered the vector AdCMV.VEGF (109 pfu) by direct myocardial injection (two injections per animal; 12 animals). Tissue samples (1 cm3) from the site of vector administration were harvested and evaluated for VEGF expression immediately and at 2, 5, 7, and 14 days after vector administration. Tissue injected with the AdCMV.CAT vector was used as a negative control.
Quantification of VEGF expression in myocardium was performed with the Quantikine human VEGF immunoassay (R&D Systems, Inc, Minneapolis, MN). Tissue samples (0.5 g) from the sites of vector administration were homogenized with protein lysis buffer (10 mmol/L Tris-HCl, pH 8, 0.14 mol/L NaCl, 0.025% NaN3, 2% Triton X-100, and 1 mmol/L phenylmethylsulfonyl fluoride; 2 mL/g tissue), protein determinations were performed, aliquots of protein lysate (100 µg) were analyzed in triplicate, and absorbance was measured at 450 nm using a microplate reader. The concentration of VEGF was normalized to mg protein. The spatial limit of VEGF expression was determined by evaluation of tissue samples from animals sacrificed at 7 days. Tissue was divided into central, peripheral, epicardial, and endocardial components as described earlier, and each sample was evaluated individually for VEGF expression.
To determine whether localized expression of VEGF would result in detectable levels of VEGF in the serum, we obtained blood samples from the animals before vector administration and at the times of sacrifice at 2, 5, 7, and 14 days after vector administration. Quantification of VEGF was performed by enzyme-linked immunosorbent assay (R&D Systems) on 50-µL samples of serum, as described earlier.
Host Response to Direct Myocardial Administration of Adenovirus Vectors
To evaluate the systemic effect of direct myocardial injections of adenovirus vectors in the myocardium, serum biochemistry and complete blood count indices were monitored over time. Blood samples for white blood cell count, hematocrit, platelet count, alkaline phosphatase, serum glutamic-pyruvic transaminase, bilirubin, and creatinine were obtained from the animals before vector administration and at 2, 7, and 14 days after vector administration. Values for each time were averaged and reported as mean ± standard error of the mean. Serum chemistry determinations were calculated with the Du Pont Analyst Benchtop Chemistry System (Du Pont Co, Wilmington, DE), and complete blood count determinations were made with the System 900 Hematology Analyzer (Serono Diagnostics, Allentown, PA).
To determine the effect of direct myocardial gene transfer on cardiac function, we performed transthoracic two-dimensional Doppler and echocardiograms using a Hewlett-Packard 2500 echocardiographic machine (Hewlett-Packard Co, Andover, MA) and a 3.5-MHz transducer. Images were recorded on standard VHS videotape, and off-line analysis was performed using the Digisonics Off-Line Analysis System (Digisonics Inc, Houston, TX). The following images were obtained preoperatively and either 5 to 7 days postoperatively or 14 days postoperatively: the parasternal long-axis view, the parasternal short-axis view at the tip of the papillary muscles, and the apical five-chamber view. Pulsed-wave Doppler echocardiography at the level of the aortic annulus was also performed from the apical five-chamber view.
Off-line analysis of regional wall thickening was performed by tracing the endocardial and epicardial surfaces of the left ventricle in both diastole and systole. The ventricle was then divided into six equal radial segments, with segment 1 beginning at the inferior ventricular septum and subsequent segments labeled consecutively in a clockwise fashion, with segment 6 ending at the inferior wall. Segments 3 and 4, therefore, represent the anterolateral free wall of the left ventricle. The mean wall thickness of each segment was determined. Systolic wall thickening in each segment was defined as the mean systolic wall thickness minus the mean diastolic wall thickness. As an assessment of global left ventricular function, cardiac output was calculated using standard Doppler-derived stroke volume (aortic annular area times the velocity time integral of the flow velocity profile across the aortic annulus) and the recorded heart rate.
Statistical Analysis
Results are presented as mean ± standard error of the mean. Statistical analysis was performed by the unpaired two-tailed Student's t test, and statistical significance was defined as p < 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). A dose response for direct myocardial adenovirus vector administration was demonstrated using the AdCMV.CAT vector. Chloramphenicol acetyl transferase activity was detected with a dose as low as 107 pfu, and increased as a function of dose to 109 pfu, 7 days after vector administration (Fig 3). Consistent with the fact that the CAT protein is not endogenous to mammalian tissue, no measurable CAT activity was detected in naive (untreated) myocardium.
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; p < 0.03, all comparisons). At all times examined, in both the epicardial and endocardial halves of the treated myocardium, CAT activity was at least twofold higher in tissue samples within 10 mm of the site of injection (central) than in tissue samples 11 to 15 mm from the site of injection (peripheral). These differences proved significant at 2 and 5 days (p < 0.04, all comparisons).
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). By day 14, VEGF levels had returned to baseline. In contrast, administration of the control vector AdCMV.CAT did not result in significantly increased levels of VEGF expression over baseline at any of the times examined (p > 0.09, all comparisons). The levels of VEGF on day 0, obtained immediately after vector administration, were similar to the levels in naive tissue, confirming that the viral preparation was not contaminated with VEGF protein (p > 0.2). Furthermore, in support of the concept that adenovirus vector delivery provides a localized gene transfer strategy, no levels of VEGF could be detected in the serum of treated animals at any of the times examined (data not shown).
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Host Response to Direct Myocardial Administration of Adenovirus Vectors
All animals that received adenovirus vectors survived to their predetermined times for sacrifice. No animals demonstrated failure to thrive or tachycardia or were febrile, and no wound infections developed. There were no significant changes over baseline in white blood cell count, hematocrit, platelet count, alkaline phosphatase, serum glutamic-pyruvic transaminase, bilirubin, or creatinine in the animals examined at 2, 7, and 14 days after vector administration (Table 1).
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). Regional wall motion assessment revealed no significant difference in the systolic wall thickening of any of the six radial segments between the preoperative and postoperative studies. Cardiac output also did not change significantly between the preoperative and postoperative studies.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The present study demonstrated that for at least 14 days after direct myocardial administration of adenovirus vectors, high levels of protein expression can be achieved in tissue as far as 1.5 cm from the site of vector administration, without significant transfection of distant organs. This direct in vivo gene transfer strategy was achieved without compromising cardiac function, as assessed by echocardiography, and without affecting the animals systemically, as assessed by physical examination and routine laboratory evaluation. This study further demonstrated that in a large animal model, myocardial expression of a therapeutic and biologically relevant gene can be accomplished using an adenovirus-mediated gene transfer strategy.
Although myocardial gene transfer has been accomplished by coronary catheterization in a rabbit model [2], there is recent evidence in a porcine model that direct myocardial injection may provide a more localized and concentrated level of gene expression [26]. Our data support this concept. Using identical doses of vector, levels of protein activity in myocardium were significantly lower in animals undergoing catheter-based delivery compared with animals receiving direct myocardial injections. An explanation for our low levels of catheter-based gene expression might be that an intracoronary delivery technique in dogs may require a higher dose of vector than anticipated. Barr and associates [2] reported successful myocardial gene transfer using the same intracoronary dose and volume that were used in our study (109 pfu; 1 mL). However, their experiments were conducted in rabbits, and vector was administered over a period of 1 minute, whereas our delivery was accomplished over 10 seconds. A slower administration, a higher dose of vector, or a different catheterization technique, such as one that uses a double balloon catheter to maintain longer vector contact, might improve localized gene transfer.
Regardless of the potential of intracoronary delivery techniques, we have demonstrated that by using an identical dose of vector with a direct myocardial injection technique, high levels of localized protein expression can be achieved for up to 14 days after vector administration. The spatial limit of gene expression achieved-up to 1.5 cm from the site of vector administration-is significantly greater than previously reported. Studies using direct myocardial gene transfer to rodent [27] and porcine [4] myocardium have documented gene transfer in tissue only as far as 5 mm from the site of injection, suggesting that a series of injections of vector close to one another would be necessary to achieve gene expression over a clinically relevant territory of myocardium. The data presented in this study, however, demonstrated that sustained gene expression can be achieved over a reasonable territory of myocardium with a single direct injection technique. An understanding of the spatial limits of gene expression will be important for proper design of future myocardial gene delivery strategies.
One potential explanation for the enhanced spatial gene expression that we detected, compared with previous studies, is that the superficial injection technique that we used (3 to 5 mm from the epicardial surface) might allow more extravasation of the vector through the epicardial surface at the time of administration. Furthermore, canine myocardium, known to have prominent epicardial vasculature, might accept and disperse a volume of 100 µL injectate more than porcine or rodent myocardium, which is known to have less extensive vasculature. Our technique of systematically analyzing individual tissue samples at specific distances from the site of vector injection might also be a more sensitive method of detecting gene expression. Finally, the use of CAT as a reporter gene, which can be quantified at very low levels of activity, may provide a more sensitive detection system than other reporter genes used previously, such as ß-galactosidase.
The administration of a number of growth factor proteins, including VEGF, has been shown previously to induce angiogenesis and revascularization in models of myocardial and peripheral vascular ischemia. Two studies in particular have demonstrated that the myocardial administration of VEGF protein in the setting of ischemia is capable of eliciting an angiogenic response. In a canine model of myocardial ischemia, daily administration of VEGF through an indwelling catheter in the distal left circumflex artery resulted in an increase in collateral blood flow to ischemic myocardium and an increase in the density of intramyocardial vessels [12]. Similarly, in a porcine model of myocardial ischemia, continuous administration of VEGF to the myocardium resulted in angiogenesis, as demonstrated by magnetic resonance imaging [13]. A consistent feature in both of these models was that myocardium was exposed to VEGF over a sustained number of days, presumably for long enough to initiate and maintain an angiogenic response.
The fact that this study has demonstrated sustained and localized levels of VEGF in myocardium after a single in vivo administration of AdCMV.VEGF suggests that an adenovirus-mediated gene transfer strategy might be capable of stimulating site-directed angiogenesis in target organs after a single vector administration. Such a strategy might prove to be less cumbersome than existing models, which require either continuous or repeated administration of the VEGF protein. Furthermore, because adenovirus-mediated direct myocardial gene transfer has been demonstrated to involve minimal transfection of distant organs, the theoretic risk of exposing sensitive nondiseased organs to VEGF would be minimized.
This study has also demonstrated the safety of adenovirus-mediated myocardial gene transfer. For at least 14 days after direct myocardial vector delivery, no significant change in global or regional left ventricular function was detected by echocardiography. Furthermore, no systemic adverse reactions were noted, as evidenced by the well-being of the animals and by stable white blood cell counts and liver function tests. Thus, adenovirus vector administration directly to the myocardium of large animals appears to be safe and well tolerated, at least as determined by the sensitivities of these assays.
In summary, the importance of this study is that a wide spatial limit of gene expression has been characterized after adenovirus-mediated direct myocardial gene transfer, and the delivery system has been shown to be safe and well tolerated. In addition, using a large animal model with physiology similar to that of a human, we showed that a single in vivo administration of an adenovirus vector encoding a therapeutic angiogenic protein (VEGF) resulted in sustained and localized protein expression for a number of days after gene transfer. The next important application of this gene transfer strategy will be to evaluate the angiogenic potential of this and other vectors when administered to normal and ischemic myocardium.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Rosengart, Department of Cardiothoracic Surgery, The New York Hospital-Cornell Medical Center, 520 E 70th St, F2100, New York, NY 10021.
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