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Ann Thorac Surg 2000;69:14-23
© 2000 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, The New York Hospital-Cornell Medical Center, New York, New York, USA
b Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, New York, New York, USA
c Division of Nuclear Medicine, The New York Hospital-Cornell Medical Center, New York, New York, USA
d Division of Cardiology, The New York Hospital-Cornell Medical Center, New York, New York, USA
Address reprint requests to Dr Rosengart, Evanston Northwestern Healthcare, 2650 Ridge Ave, Evanston, IL 60201
e-mail: t-rosengart{at}nwu.edu
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract |
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Methods. Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial AdGVVEGF121.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model.
Results. Intramyocardial delivery of AdGVVEGF121.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial AdGVVEGF121.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial AdGVVEGF121.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration.
Conclusions. These studies demonstrate that direct intramyocardial administration of AdGVVEGF121.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human trials of gene therapy-mediated cardiac angiogenesis.
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Introduction |
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One of the most widely studied angiogens is vascular endothelial growth factor (VEGF), a homodimeric 34- to 46-kDa heparin-binding glycoprotein [8]. As a result of posttranscriptional mRNA splicing, VEGF normally exists as four isoforms of 121-, 165-, 189-, and 206-amino acid residues [9]. Structurally related genes, designated VEGF-B, C, D, E, and F, have also recently been identified [10–12]. VEGF is one of the most specific angiogens due to localization of its receptors almost exclusively to vascular endothelial cells [13]. Because of this selectivity, VEGF has attracted much interest as a therapeutic angiogen, allowing stimulation of new blood vessel formation while theoretically minimizing potential risks of fibrosis or intimal hyperplasia due to promiscuous stimulation or replication of fibroblasts or smooth muscle cells by angiogens that do not have the endothelial selectivity of VEGF.
Gene therapy for angiogenesis is a delivery strategy in which the cDNA for a selected angiogen, such as VEGF, is delivered to the target tissue. This strategy potentially allows prolonged, localized angiogen expression sufficient to induce angiogenesis and thereby provide "biological revascularization" of ischemic tissues, but avoids the potential toxicity associated with excessive or systemic angiogen dissemination [4, 7, 14, 15]. We have previously reported that the use of an adenovirus (Ad) vector coding for the VEGF 121 cDNA (AdGVVEGF121.10) yields improved myocardial perfusion, contractile function, and angiographically identified collateral vessels in a porcine model of myocardial ischemia [7].
In the present study, we have capitalized on the adaptation of the new TaqMan real-time quantitative polymerase chain reaction (PCR) technology to compare the distribution of the AdGVVEGF121.10 vector after intramyocardial vs intracoronary administration, and we have compared the tissue levels of VEGF resulting from these alternative delivery strategies. Based on the striking focal distribution of the genome of the AdGVVEGF121.10 vector and VEGF levels in the injected area after direct intramyocardial administration, we have reassessed the focality of collateral vessel development and improved cardiac perfusion in our prior study of the ischemic porcine myocardium [7]. The findings of this analysis, together with the quantitative demonstration of the highly focal nature of intramyocardial administration of AdGVVEGF121.10 angiogen, strongly support the strategy of direct intramyocardial administration of an Ad vector coding for VEGF121 to induce focal myocardial therapeutic angiogenesis in individuals with severe coronary artery disease.
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Material and methods |
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Intramyocardial vector delivery in the rat
To assess the persistence of AdGVVEGF121.10 vector genome and levels of expressed VEGF protein, Sprague-Dawley rats (250 to 350 g; Charles River Laboratories, Wilmington, MA or Taconic Farms, Germantown, NY) underwent left parasternotomy, with the assistance of a rodent ventilator (Harvard Apparatus Inc, South Natick, MA), and apical myocardial injection of 1010 particle units (pu; 50 µL) of AdGVVEGF121.10 or the AdNull control vector diluted with phosphate-buffered saline, pH 7.4 (PBS). The animals were sacrificed at varying time points and the apex was homogenized (see below) with protein lysis buffer and subjected to TaqMan real-time quantitative PCR assessment and ELISA, to assess vector genome and VEGF protein levels, respectively (see below).
Porcine model of intramyocardial vs. intracoronary ad vector administration
To compare the effectiveness of intracoronary vs. intramyocardial Ad vector administration, castrated, male Yorkshire swine (28 to 30 kg; Tom Morris Farms, Wetherstown, MD) underwent left thoracotomy and administration of AdGVVEGF121.10 via direct intramyocardial injection, at 10 sites in the circumflex distribution (total dose 1012 pu, total volume 1,000 µL; each of 10 sites receiving 1011 pu in 100 µL PBS per injection), or intracoronary (circumflex artery) injection as 1012 pu in 1,000 µL PBS over 20 seconds. The sites of intramyocardial injection were marked with India ink (Eberhard Faber, Inc, Lewisburg, TN) for subsequent identification. Similar identification marks were made in the myocardium of the circumflex territory of pigs receiving intracoronary injections to standardize the regions from which subsequent samples were taken. For each method of administration, the pigs (n = 3, each method) were sacrificed 1 h after vector administration for evaluation of regional myocardial Ad vector genome by TaqMan analysis, and the remaining pigs (n = 6) were sacrificed 1 day after vector administration for analysis of regional myocardial VEGF levels by ELISA. At the time of sacrifice of each pig, 10 biopsies (1.0 to 1.5 cm diameter) were taken from the left ventricular circumflex territory around the previously marked sites, effectively dividing the entire circumflex territory into 10 samples, and two samples each were taken from the mid-right ventricle and from the mid-septum (1.0 to 1.5 cm diameter, each sample).
Tissue ad vector genome levels
To evaluate Ad vector genome levels, tissue biopsies obtained as described above were analyzed utilizing real-time quantitative PCR and the TaqMan chemistry [17]. The samples were immediately homogenized with protein lysis buffer (10 mM Tris-HCl, pH 8, 0.14 M NaCl, 0.025% NaN3, 2% Triton X-100, and 1 mM phenylmethylsulfonyl chloride; 2 mL/g tissue) before DNA extraction. DNA was extracted from the homogenate samples using the QIamp tissue kit (QIAGEN Inc, Santa Clarita, CA). Triplicate PCR reactions (50 µL each) were established with 5 µL of both 1:10 and 1:100 dilutions of the DNA in water. Amplification reactions were prepared using reagents from Perkin Elmer (Foster City, CA) and contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01 mM ethylenediaminetetraacetate acid, 60 nM 5-carboxyrhodamine, 3 mM MgCl2, 200 nM each dCTP, dGTP, and dATP, 400 nM dUTP, 0.5 units urical N-glycosylase, (UNGase) 0.25 units AmpliTaq Gold DNA polymerase, 200 nM forward primer (5'-AATAAACAAGTTCCCGGATCGAT), 200 nM reverse primer (5'-GCACATAGGAGAGATGAGCTTCC), and 100 nM TaqMan central probe (5'-CCGCCTCGGCTTGTCACATTTTTC). The TaqMan probe was labeled with the reporter group FAM (6-carboxy fluorescein) on the 5' end and the quencher TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) on the 3' end. The primer and probe combination was designed using the PrimerExpress software (Perkin Elmer) and was directed towards the transgene/promoter junction region of the AdGVVEGF121.10 vector [7]. The forward primer overlaps the cytomegalovirus (CMV) promoter while the probe and reverse primer are complementary to the VEGF121 cDNA. This design was chosen to ensure that the endogenous VEGF gene of any species, which would be adjacent to its own promoter rather than the CMV promoter (the promoter used in the vector expression cassette), would not be amplified by the chosen primer set. To construct a standard curve, the expression plasmid in which CMV drives transcription of the VEGF121 cDNA originally used to construct the Ad vector was used. Samples (standard or unknown) were amplified for 40 cycles in a Perkin Elmer 7700 sequence detection system after 10 minutes at 50°C for UNGase to degrade carryover contamination, and 10 minutes at 94°C to activate the AmpliTaq Gold. Cycling conditions were 15 seconds at 94°C followed by 1 minute at 60°C with continuous monitoring of the fluorescence. Data were processed by the SDS 1.6 software (Perkin Elmer) to generate standard curves and to determine the concentration of target in the unknowns by interpolation. A 25% difference in the vector amount adjusted for dilution in the 1:10 and 1:100 dilutions of the DNA was considered acceptable. Total DNA concentration was determined by measuring the A260 of dilutions of the DNA stocks. The vector levels from the TaqMan experiments were averaged and normalized to rat or pig genome level using the approximation that each diploid cell contains 6.6 pg of DNA. The mean Ad genome/cell genome value were then calculated for each tissue sample.
Tissue VEGF levels
Tissue samples obtained for analysis of VEGF protein levels were immediately homogenized with protein lysis buffer, protein determinations were performed (Pierce, Rockford, IL), aliquots of protein lysate were analyzed in duplicate, and absorbance was measured at 450 nm using a microplate reader. The standard wells against which the samples were compared contained equal amounts of normal pig heart homogenate to correct for any background that might interfere with the absorbance. The concentration of VEGF was normalized to milligrams protein, and the mean value for the tissue sample was calculated. The lower limit of VEGF protein detection by this assay was 5.0 pg/mg of protein.
Porcine model of myocardial ischemia
Based on the striking focal localization of the Ad genome and VEGF levels in the myocardium (see Results), we reassessed data previously acquired in a porcine model of myocardial ischemia [7]. Further details of the model can be found in the original description of these animals in the study by Mack and associates [7]. The pigs were randomized to receive 10 injections (100 µL each) of the therapeutic vector AdGVVEGF121.10 (total dose 109 plaque-forming units [pfu]; n = 8) or the control vector AdNull (total dose 109 pfu; n = 7) injected at 10 sites in the circumflex distribution using a 0.5 mL U-100 insulin syringe with a 28-gauge needle (Beckton Dickinson & Co, Franklin Lakes, NJ).
At the time of sacrifice (28 days after vector administration), the hearts were perfusion fixed, and ex vivo coronary angiography was performed in a blinded manner [7]. Prior analysis of these angiograms demonstrated significant development of collateral vessels in the pigs treated with AdGVVEGF121.10 compared with those receiving the control AdNull vector [7]. For the present study, the angiograms were reassessed to evaluate focal parameters of collateral development, including: (1) an index of collateral flow to the circumflex artery, quantified in a blinded fashion as described by Gibson and associates [18], and (2) the presence and location of a focal vascular plexus. The frame at which contrast initially filled the left anterior descending artery was assigned as frame 0. The cinefilm was then advanced until contrast was seen to initially fill the circumflex or obtuse marginal arteries, and the number of frames advanced to this stage was recorded. The presence or absence and the location of a vascular plexus (defined as at least three collateral vessels per centimeter) within or outside the region of vector administration was also analyzed for each pig.
Regional myocardial perfusion was assessed during rest and stress (rapid atrial pacing 200 beats/min) at the time of vector administration (day 0) and 28 days later utilizing 99mTc-sestamibi (Cardiolite; DuPont Pharma, N. Billerica, MA) and single photon emission computed tomography (SPECT) as previously described [7]. Prior analysis of these scans demonstrated significant improvement of blood flow with stress in the circumflex distribution with AdGVVEGF121.10 compared with AdNull animals [7]. For the present study, these scans were reassessed to evaluate the focal nature (circumflex distribution vs. septum) of the improvement in flow. To accomplish this, a multi-level semiquantitative visual interpretation of the SPECT images was performed on a subset of AdGVVEGF121.10 (n = 6) and AdNull (n = 4) pigs at both time points, as described by Hachamovitch and associates [19]. Three short-axis views were analyzed at rest and stress, at days 0 and 28 postvector, for each pig. Each segment was scored, in a blinded fashion, on a scale from 0 to 4 (0 = absent, 1 = severe reduction, 2 = moderate, 3 = equivocal, 4 = normal radioisotope uptake). The two contiguous sections with the best score (corresponding to the septum in all cases), and the two contiguous sections from each of the three levels with the worst score (corresponding to the circumflex territory in all cases), from rest and stress imaging were added together to generate, respectively, a summed rest and summed stress score for the septum and circumflex territories (0 = no perfusion, 24 = normal perfusion). The differences between these scores were calculated for the septum and circumflex territory for each pig at each time point. This "summed difference" score ("regional ischemia score") provides a measure of the extent and the severity of ischemia in each of these territories (0 = no ischemia, 24 = maximal ischemia).
Statistical analysis
Statistical analyses were performed with the 
2 test for categorical data and analysis of variance or Student’s t tests for continuous data, as appropriate. A paired Student’s t test was used for comparison of the same group at different time points. The Mann-Whitney nonparametric test was used if the data were not normally distributed. All values reported were expressed as the mean ± the standard error of the mean.
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Results |
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Comment |
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Angiogen delivery options: choice of vector
A number of experimental animal studies have demonstrated that delivery of angiogens to the ischemic myocardium will improve blood flow and viability of the heart [2, 4, 5]. Many of these strategies have utilized some means of sustained angiogen delivery, including somewhat cumbersome catheter/reservoir-based and continuous infusion techniques, or the use of angiogen-impregnated sustained release polymers or biologic substrates [6]. Other strategies have utilized one-time intravascular administration of a protein angiogen, but these strategies have been associated with significant systemic adverse effects, such as the hypotension associated with VEGF administration [20], and the theoretical risk of promiscuous angiogenesis elsewhere in the body.
With the confirmation of the capability to induce some degree of angiogenesis in these studies, it is important to determine a means of optimally inducing localized angiogenesis with minimal angiogen dissemination, thereby reducing potential remote toxicity. Gene therapy strategies, whereby the single administration of an appropriate angiogen induces the sustained expression of the relevant gene product after transfection of the target cells at the site of ischemia, may represent an ideal means of providing biologic revascularization.
Our choice of an Ad vector-mediated delivery strategy for the treatment of cardiac ischemia is based on the specific characteristics of Ad vectors in regard to efficiency of vector delivery to the myocardium and to vector pharmacokinetics. Ad vectors are 103- to 104-fold more efficient in vivo (per transgene) at transfecting target cells than plasmid systems, and do not require replicating cells for transfection, as is the case with retroviral vectors [21]. Because the Ad vector genome remains in an epichromosomal position after transfection without integration into the host genome, the use of an Ad vector avoids the theoretical risk of host cell mutagenesis, which may be a concern with vectors that integrate into the host genome, such as retroviral and adeno-associated virus vectors [21, 22]. For the angiogenic therapy of ischemic heart disease, the goal is to produce sufficient collateral blood vessels to normalize blood flow to the ischemic territory, but not to produce pathologic angiogenesis, and not to generate collaterals in regions of the myocardium where they are not needed. In this regard, studies in experimental animals, and our human experience using Ad vectors to treat cystic fibrosis, have demonstrated that the Ad vectors of the design used in the present study will very effectively express a transgene in an internal organ, but the vector genome is progressively lost, limiting transgene expression to 1 to 2 weeks [14, 23]. While this poses a challenge for the treatment of hereditary disorders where persistent expression is necessary, it is ideal for the purposes of therapeutic angiogenesis, where induction of therapeutic angiogenesis requires only a limited duration of expression of the therapeutic gene. In this context, recent studies by Springer and associates [24] have shown that prolonged expression of the VEGF cDNA in skeletal muscle induces the formation of hemangiomas. Finally, an added advantage of the Ad vector is that the high affinity of the Ad vector for its receptor on the cell’s surface helps to localize the vector to the area of administration.
The mechanism that limits Ad vector persistence in the myocardium is not known. While anti-Ad cellular immune mechanisms may play a role [23], studies in pigs with myocardial ischemia show that administration of the AdGVVEGF121.10 vector is likely not associated with significant inflammation nor necrosis of the myocardium [25], suggesting that the mechanisms of vector elimination are complex, and not associated with significant loss of the myocardial cells to which the vector genome has been transferred.
Angiogen delivery options: choice of route
To date, two methods of administration have been utilized to deliver an Ad vector coding for an angiogenic gene to the ischemic myocardium: intracoronary and intramyocardial. The intracoronary strategy has been utilized in a porcine model of myocardial ischemia to deliver an Ad vector coding for the fibroblast growth factor-5 cDNA [4], and direct myocardial administration has been employed in a similar model to deliver an Ad vector coding for the VEGF121 cDNA [7]. Both methods have demonstrated efficacy in terms of myocardial revascularization, and both are feasible techniques for utilization in individuals with coronary artery disease.
The present study demonstrates that vector administration directly to porcine myocardium results in far superior vector genome localization in the treated myocardium than does intracoronary injection of the Ad vector. This in turn leads to greater localized VEGF expression, as evidenced by the greater local VEGF levels after intramyocardial versus intracoronary administration of angiogen. In this context, for the same amount of vector, direct myocardial administration provides markedly higher levels of VEGF where it is needed, and limits the VEGF expression to the areas where new collaterals develop. Although it is not known what minimal myocardial concentration of an angiogen will translate into clinical efficacy, it appears that intramyocardial as opposed to intracoronary delivery represents a means of limiting the total dose used, as well as focalizing angiogenic therapy, and thus potentially avoiding promiscuous angiogenesis at other sites [26].
Characterization of focal angiogenesis mediated by intramyocardial administration of the AdGVVEGF121.10 angiogen
Direct administration of an Ad vector coding for the cDNA for VEGF121 to the ischemic porcine myocardium induces the development of a plexus of collateral vessels localized to the region of vector delivery, with an associated brisk reconstitution of blood flow to the territory of an experimentally occluded coronary artery. Perfusion scans confirm that this biologic revascularization improves blood flow during myocardial stress throughout the ischemic territory. Importantly, the observation of persistence of normal perfusion in the left anterior descending territory, the source of collateral flow to the circumflex territory, suggests that biological revascularization does not induce a "steal" phenomenon rendering ischemic the myocardial territory that is the source of collateral flow. The near-normalization of blood flow and myocardial function demonstrated in this, and our previous analysis [7] suggests that complete reconstitution of flow is possible with angiogenic therapy, and that intramyocardial AdGVVEGF121.10 delivery may represent an optimal strategy to accomplish this in the clinical setting.
In conclusion, the primary significance of the observations in the present study is that direct myocardial Ad vector administration produces greater regional myocardial Ad vector genome localization than intracoronary administration. Consequently, transgene expression follows a similar pattern, resulting in high local VEGF concentrations of the transgene product. Such a strategy induces angiogenesis on a scale that nearly normalizes blood flow. These findings may be of importance in considering human trials of gene therapy-mediated cardiac angiogenesis.
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