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J Thorac Cardiovasc Surg 1998;115:168-177
© 1998 Mosby, Inc.
CARDIOPULMONARY SUPPORT AND PHYSIOLOGY |
From the Department of Cardiothoracic Surgery,a Division of Pulmonary and Critical Care Medicine,b Division of Nuclear Medicine,c and Division of Cardiology,d The New York Hospital–Cornell Medical Center, New York, N.Y., and GenVec, Inc.,e Rockville, Md. These studies were supported, in part, by The Jeffry M. and Barbara Picower Foundation, Palm Beach, Florida; by GenVec, Inc., Rockville, Maryland; the Will Rogers Memorial Fund, White Plains, New York; The New York Heart Association, Grant-in-Aid 94066; and GCRC Grant RR00102.
Read at the Seventy-seventh Annual Meeting of The American Association for Thoracic Surgery, Washington, D.C., May 4-7, 1997.
Received for publication May 9, 1997. Accepted for publication Sept. 15, 1997. Revisions requested July 15, 1997; revisions received Sept. 2, 1997. Address for reprints: The New York Hospital–Cornell Medical Center, New York, NY 10021.
Abstract
Objectives: Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, can be delivered to targeted tissues by means of a replication-deficient adenovirus (Ad) vector. We hypothesized that direct administration of Ad vector expressing the VEGF121 complementary deoxyribonucleic acid (AdGVVEGF121.10) into regions of ischemic myocardium would enhance collateral vessel formation and improve regional perfusion and function.
Methods: Yorkshire swine underwent thoracotomy and placement of an Ameroid constrictor (Research Instruments & MFG, Corvallis, Ore.) on the circumflex coronary artery. Three weeks later, myocardial perfusion and function were assessed by single photon emission computed tomography imaging (SPECT) with99mTc-labeled sestamibi and by echocardiography during rest and stress. AdGVVEGF121.10 (n = 7) or the control vector, AdNull (n = 8), was administered directly into the myocardium at 10 sites in the circumflex distribution (10
8 pfu/site). Four weeks later, these studies were repeated and ex vivo angiography was performed.
Results: SPECT imaging 4 weeks after vector administration demonstrated significant reduction in the ischemic area at stress in AdGVVEFG121.10-treated animals compared with AdNull control animals (p = 0.005). Stress echocardiography at the same time demonstrated improved segmental wall thickening in AdGVVEGF121.10 animals compared with AdNull control animals (p = 0.03), with AdGVVEGF121.10 animals showing nearly normalized function in the circumflex distribution. Collateral vessel development assessed by angiography was also significantly greater in AdGVVEGF121.10 animals than in AdNull control animals (p = 0.04), with almost complete reconstitution of circumflex filling in AdGVVEGF121.10 animals.
Conclusions: An Ad vector expressing the VEGF121 cDNA induces collateral vessel development in ischemic myocardium and results in significant improvement in both myocardial perfusion and function. Such a strategy may be useful in patients with ischemic heart disease in whom complete revascularization is not possible.
Despite continued advances in the treatment of ischemic heart disease, a large population of individuals with diffuse coronary artery disease exists for whom conventional therapies such as percutaneous angioplasty and bypass surgery provide little or no benefit. A new strategy that may be applicable to such patients is "therapeutic angiogenesis," the induction of new blood vessel development into areas with limited blood flow. 1,2 Therapeutic angiogenesis capitalizes on the discovery of protein mediators that elicit angiogenesis, a complex process that includes the migration and proliferation of endothelial cells, vascular tube formation, and linkage to the preexisting vascular network. 3,4 Several of these protein mediators have been shown to promote revascularization of ischemic tissues in models of myocardial or peripheral ischemia. 1-3,5–9
Vascular endothelial growth factor (VEGF), a homodimeric 34 to 46 kDa heparin-binding glycoprotein, is the most specific of the known angiogenic mediators because of localization of its receptors almost exclusively on endothelial cells. 10–12 The human VEGF gene is expressed as four isoforms secondary to posttranscriptional splicing, producing proteins of 121, 165, 189, and 206 residues. The observations that VEGF and VEGF receptors (flk-1/KDR and flt-1) are up-regulated under ischemic conditions is consistent with the concept that VEGF is an endogenous mediator of angiogenesis. 11–13
Gene therapy, in which the complementary deoxyribonucleic acid (cDNA) for VEGF is delivered directly to tissues, has the potential to induce localized VEGF expression that is of limited duration, thus avoiding the toxicity and promiscuous angiogenesis potentially associated with systemic protein therapy. 14–18 In examining this hypothesis, the present study uses a porcine myocardial ischemia model to demonstrate that direct administration of a replication-deficient adenovirus (Ad) vector coding for the human VEGF121 cDNA (AdGVVEGF121.10) into ischemic myocardium induces a "biologic bypass"—collateralization around a site of coronary occlusion, with concomitant improvement in regional myocardial perfusion and function during stress-induced myocardial ischemia.
Methods
Experimental model of myocardial ischemia.
A model of chronic myocardial ischemia was created in Yorkshire swine (28 to 30 kg). All animal care procedures were in accordance with institutional guidelines. Animals were sedated with intramuscular tiletamine and zolazepam (Telazol, 3.3 mg/kg) and xylazine (0.10 mg/kg), intubated, and sedation was maintained with 0.5% to 2.0% isoflurane. A limited left thoracotomy was performed in a sterile fashion through the fifth intercostal space and a small incision was made in the pericardium. A 2.5 mm internal diameter Ameroid constrictor (Research Instruments & MFG, Corvallis, Ore.) was placed around the circumflex artery as proximally as possible. Topical lidocaine 1% solution was applied to the circumflex artery at the Ameroid constrictor site to prevent coronary artery spasm. The pericardium and chest were then closed and the animal was allowed to recover.
Ad vectors.
The replication-deficient vector AdGV-VEGF121.10 is an E1a-, partial E1b-, partial E3–Ad vector that contains an expression cassette in the E1 position (right to left) containing the cytomegalovirus immediate early promoter/enhancer, an artificial splice sequence, the human VEGF121 cDNA, and the SV40 polyA/stop signal. AdNull (similar to AdGVVEGF121.10, but with no gene in the expression cassette) was used as a control vector. 19 All Ad vectors were propagated and titrated in 293 cells, purified by cesium chloride density purification, dialyzed, and stored at –70° C 20. The viral stocks were demonstrated to be free of replication-competent wild type Ad. Biologic activity of the VEGF121 transgene product was confirmed by demonstrating proliferation of human umbilical vein endothelial cells using [3H]thymidine incorporation, and in vivo confirmation of transgene expression was determined by enzyme-linked immunosorbent assay analysis of myocardial biopsy specimens obtained from AdVEGF121.10 injection sites 3 days after vector administration. 17
In vivo administration of Ad vectors.
Three weeks after Ameroid constrictor placement, the left thoracotomy was reopened and administration of the therapeutic vector, AdGVVEGF121.10, or the control vector, AdNull, was performed by direct myocardial injection (Fig. 1). Each vector was injected at 10 sites, each in 100 µl phosphate-buffered saline solution, pH 7.4, in the circumflex distribution (108 pfu/injection). Pacing wires were placed in the left atrial appendage and tunneled subcutaneously for subsequent stress technetium 99m (99mTc)-labeled sestamibi (Cardiolite, DuPont Pharma, N. Billerica, Mass.) assessment of regional myocardial perfusion by single photon emission computed tomography (SPECT) and echocardiographic assessment of regional wall thickening.
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The rest and stress SPECT studies were processed in a blinded fashion with the use of an integrated ADAC Pegasys computer. Stress and rest circumferential count profiles (polar plots) at the midventricular level were constructed by dividing the midventricular short-axis image into 60 angular segments centered on the ventricular cavity, determining the number of counts per segment, normalizing the number of counts in each segment to the segment with the maximum number of counts (assigned a reference value of 100), and plotting the normalized counts per segment versus the angular position of the segment. The polar plots were transferred to ASCII files for further analysis with the program SIGMAPLOT (Jandel Scientific, Corte Madera, Calif.).
For each animal, the extent of myocardial ischemia ("area") was determined from the difference between the rest and stress polar plots. The maximum severity of ischemia ("ischemia maximum") in the circumflex distribution was determined by ascertaining the point of greatest difference between the rest and stress plots and measuring the difference in the plots at that point. The percent improvement in myocardial perfusion for each animal was calculated for these two parameters as ("parameter" at 3 weeks - "parameter" at 7 weeks x 100)/("parameter" at 3 weeks).
Echocardiographic assessment of regional myocardial function.
Baseline regional myocardial function was assessed by echocardiography at rest and during stress at the time of vector administration. Animals were sedated and placed in the left lateral decubitus position, and standard two-dimensional and M-mode transthoracic images were obtained with an HP2500 echocardiographic machine and a 3.0/3.5 MHz dual-frequency transthoracic transducer (Hewlett-Packard, Andover, Mass.). From the right parasternal approach, short-axis, midpapillary views were obtained at rest for 3 minutes. The animals then underwent rapid left atrial pacing in a stepwise fashion to the target ventricular rate of 200 beats/min, at which time imaging was recorded for an additional 3 minutes.
Regional wall thickening was determined by a single experienced investigator in a blinded fashion, tracing the endocardial and epicardial surfaces of the left ventricle in both diastole and systole using a Digisonics CardioRevue System (Digisonics Inc., Houston, Tex.). Systolic wall thickening in each of six equal radial 60-degree segments was defined as mean systolic wall thickness - mean diastolic wall thickness. Fractional wall thickening was calculated as mean systolic wall thickening/mean diastolic wall thickness. The ischemic and nonischemic zones for each animal were defined from rapid atrial pacing images at 3 weeks (baseline ischemia) as the two contiguous segments with the lowest and highest fractional wall thickening, respectively. This corresponded in all cases with the circumflex region and the septum, respectively. The same zones for each animal were analyzed in rapid atrial pacing images at 7 weeks.
Ex vivo coronary angiography.
When each animal was put to death (4 weeks after vector administration), the heart was arrested with 40 mEq of KCl and then perfusion-fixed at 100 mm Hg with 1 L of McDowell-Trump fixative (4% formaldehyde, 1% glutaraldehyde, 1% NaH2PO4 and 0.3% NaOH adjusted to pH 7.2). Ex vivo coronary angiography was performed by the same angiographer in a blinded fashion using a 5F end-hole wedge balloon catheter (Arrow Inc., Reading, Pa.) placed in the left main coronary artery. By means of cinefluoroscopy in the standard right anterior oblique projection with continuous image acquisition, 5 ml of contrast medium (Hypaque-76, Nycomed Inc., New York, N.Y.) was injected at a continuous rate until the entire left anterior descending coronary artery and its branches were completely opacified. 21 Collateral vessels from the left anterior descending coronary artery, which reconstituted the circumflex coronary artery or obtuse marginal branch of the circumflex coronary artery, were quantified by three blinded observers using the grading method of Rentrop and associates 22 as follows: 0 = no filling of collateral vessels; 1 = filling of collateral branches of the circumflex or obtuse marginal branch without visualization of the epicardial segment; 2 and 3 = partial or complete filling of the epicardial segment of the circumflex or obtuse marginal artery via collateral vessels, respectively.
Histologic evaluation.
After angiography, the left ventricle of each heart was sectioned into three rings in the short axis. Forty 5 µm histologic sections from each heart were taken at equidistant intervals around the basal and midventricular rings, processed through paraffin, and stained with hematoxylin and eosin. Histologic evidence of infarction and inflammation for each tissue section was graded by a pathologist blinded to treatment on a scale of 0 to 4 as follows: 0 = none; 1 = one to three small areas involved; 2 = less than 10% section surface; 3 = more than 10% up to 50% section surface; and 4 = more than 50% section surface.
Statistical analysis.
Treatment was assigned in an alternating consecutive fashion to a total enrollment of 15 animals that could be evaluated for efficacy, with myocardial ischemia area defined as the primary outcome variable. Statistical analysis was carried out by means of the Mann-Whitney nonparametric test. All results are expressed as mean ± standard error of the mean.
Results
Overall assessment.
All of the 19 animals entered into the study (AdGVVEGF121.10, n = 9; AdNull, n = 10) survived until put to death 7 weeks after placement of the Ameroid constrictor, without clinical evidence of toxicity. At 3 weeks (i.e., before therapy), four of the 19 pigs (AdGVVEGF121.10, n = 2; AdNull treated, n = 2) had evidence of myocardial infarction in the circumflex region, as demonstrated by (1) a fixed defect (no difference between rest and stress) in the circumflex zone of the 99mTc-sestamibi SPECT images and (2) a thinned, akinetic posterolateral region of the left ventricle in short-axis views during echocardiography at rest. Consistent with the 99mTc-sestamibi SPECT and echocardiography suggesting myocardial infarction 3 weeks after Ameroid constrictor placement, the gross pathologic evaluation 4 weeks later showed myocardial scarring and thinning of at least 25% of the total ventricular mass. All four pigs in this subgroup also had histologic evidence of large transmural infarction. On the basis of these data, these four animals were excluded from further analysis. Thus the group of animals evaluated for efficacy of therapy included seven AdGVVEGF121.10-treated animals and eight AdNull (control) animals.
In vivo expression of the AdGVVEGF.10 vector was confirmed by demonstrating local myocardial VEGF expression after myocardial injection of 108 pfu of AdGVVEGF121.10 (n = 3). Three days after administration of the vector, myocardial levels were 0.75 ± 0.25 ng/mg protein, compared with background expression in AdNull-treated animals (p = 0.01).
AdGVVEGF121.10-mediated increase in myocardial perfusion.
Circumferential count profiles (polar plots) of 99mTc-sestamibi SPECT data from the midventricular level were used to quantify (1) the extent and severity of ischemia ("area") and (2) the most severe ischemia ("ischemia maximum") (Fig. 2, A). Circumferential plots of rest images obtained at 3 weeks typically demonstrated minimal perfusion defects, compared with plots of stress (pacing) images, which revealed decreased perfusion in the posterolateral region, corresponding to the occluded circumflex coronary artery distribution (Fig. 2, B and D). The ischemic area and ischemia maximum were characteristically unchanged from baseline in AdNull animals assessed 4 weeks after vector administration (Fig. 2, B and C). In contrast, AdGVVEGF121.10 animals demonstrated improvement in myocardial perfusion 4 weeks after vector administration, as demonstrated by decreases in the ischemic area and ischemic maximum compared with baseline (Fig. 2, D and E). Corresponding changes were noted at the apical, midventricular, and basal levels.
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The ischemia maximum in the circumflex distribution was also the same for the AdGVVEGF121.10 animals and AdNull control animals at 3 weeks (Table 1). In contrast, 4 weeks after vector administration, the ischemia maximum was significantly decreased in the AdGVVEGF121.10 animals than in the AdNull control animals. Similarly, the "percent improvement" in the ischemia maximum was 2.5-fold greater in AdGVVEGF121.10 animals than in the AdNull control animals (56% ± 8% vs 22% ± 6%, p = 0.01).
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(Fig. 3, A). In contrast, animals that received AdGVVEGF121.10 typically demonstrated nearly complete reconstitution of both the obtuse marginal and circumflex coronary circulations (Fig. 3, B and C).
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(Table 3).
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Discussion
This study demonstrates that Ad-mediated transfer of the cDNA of human VEGF isoform 121 directly into the myocardium of Yorkshire swine with an occluded circumflex coronary artery results in significant and physiologically relevant improvement in regional myocardial perfusion and contractile function during stress-induced myocardial ischemia. Importantly, this improvement was associated with increased myocardial collateral vessel development, "biologically bypassing" the experimentally occluded coronary artery segment.
VEGF as a mediator of therapeutic angiogenesis.
Considerable information is available suggesting that the VEGF protein can induce angiogenesis in a variety of animal models of ischemia. 5–9,15 In regard to cardiac ischemia, a single intracoronary bolus of VEGF165 improves blood flow to the ischemic region, 9 and continuous infusion of the VEGF165 protein via indwelling catheters increases collateral blood flow to ischemic myocardium and the numeric density of intramyocardial distribution vessels. 5 Finally, continuous administration of VEGF165 to the surface of the myocardium over 6 weeks reduces the ischemic zone and improves ejection fraction and regional myocardial wall thickening as assessed by magnetic resonance imaging. 8
Our choice of VEGF for this study is based on these findings and the endothelial cell specificity of VEGF, which offers potential advantages over the use of other growth factors with angiogenic properties (such as the fibroblast growth factors), which are also mitogenic for cells such as fibroblasts and smooth muscle cells, and thus theoretically impose the risk of unwanted fibrosis or smooth muscle cell hyperplasia. 3,23,24 Although the major VEGF isoforms appear to be equipotent in angiogenic potential, our choice of the cDNA coding for the 121 amino acid isoform of VEGF was based on the knowledge that the VEGF121 isoform does not bind heparin and thus may diffuse throughout the myocardium more readily than the other isoforms. 11,12
Ad-mediated gene transfer as a strategy for VEGF-related therapeutic angiogenesis.
Gene therapy holds several potential advantages over a protein-based strategy for therapeutic angiogenesis. Gene transfer provides the equivalent of a "slow-release depot," providing high concentrations of the therapeutic protein for a relatively extended period. Ad vector expressing VEGF provides myocardial expression of VEGF protein for up to 7 days. 17 In contrast, the VEGF protein has a very short biologic half-life in the circulation, 6 and most previous studies have consequently required continuous infusions or repetitive dosing of the growth factor to achieve a therapeutic effect. 5,8,15 Another potential advantage of gene therapy over protein-mediated angiogenic therapy is that gene transfer can be strategized to provide delivery of a high concentration of VEGF localized to the ischemic sites. In contrast, systemic administration of a single large bolus of VEGF protein can cause hypotension, and systemic therapy carries the theoretic risk of inducing inappropriate angiogenesis at sites of vascular derangement, or at sites where angiogenesis might have major adverse consequences, such as the retina, the synovium, and in occult tumors. 3,9,11,12,16
Although three gene transfer systems—naked plasmids, herpes simplex virus, and Ad—have been used to induce angiogenesis with VEGF in experimental models, we have focused on Ad vectors because their inherent properties make them ideal for use in therapeutic angiogenesis. Ad vectors achieve gene transfer in both dividing and nondividing cells, with high levels of protein expression in cardiovascular relevant sites, such as myocardium, vascular endothelium, and skeletal muscle. 14,17,25,26 The new gene transferred by an Ad vector functions in an epi-chromosomal position and thus carries little risk of inappropriately inserting the newly transferred gene in a critical site of the genome. 27 Most important, whereas long-term expression of angiogenic proteins might induce excessive, disorganized blood vessel formation, Ad vectors are highly efficient at achieving high levels of gene expression in cardiovascular tissues for only 1 to 2 weeks, thus limiting expression to that necessary to induce angiogenesis. 14,17,18 Consistent with the concept that Ad vectors can be used to deliver genes relevant to inducing myocardial angiogenesis, an Ad vector has been used to deliver the fibroblast growth factor–5 cDNA to the ischemic porcine myocardium, with resulting increases in perfusion, function, and histologic evidence of angiogenesis. 14
Host immune response is a potential disadvantage to the use of Ad vectors in some gene transfer applications. With the use of clinical grade vectors, as in the present study, however, inflammation does not appear to be a significant problem. The cellular immune response that likely limits vector expression is actually an advantage for the application of therapeutic angiogenesis, as previously discussed. Finally, although it is unlikely that repeated administrations will be needed, the theoretic development of neutralizing antibodies directed against the Ad vector that might limit the effectiveness of repeated vector administrations can likely be overcome by the use of Ad vectors of different serotypes or by immunosuppressive therapy. 28
Ad-mediated angiogenesis as a biologic revascularization strategy.
The primary significance of the present results is that a one-time direct myocardial administration of an Ad vector containing the VEGF121 cDNA induces the development of collateral vessels adequate to enhance myocardial perfusion and ameliorate regional myocardial contractile dysfunction in the setting of stress-induced myocardial ischemia. The observation in control animals of some collateral vessel development is consistent with evidence of the limited, incomplete nature of the endogenous processes of angiogenesis and collateralization that are known to occur in the setting of ischemia. In this context, the therapeutic angiogenesis described in this study can be considered as a logical enhancement of the normal, endogenous response to ischemia. With the evidence that Ad vectors are capable of delivering new genetic information safely to human beings in vivo, 27 the clinical applications of this strategy include its use as a novel therapeutic "biologic bypass" in patients with coronary disease.
We thank Emily Cogger, Kurt Budenbender, Tina Grasso, Bari Bialos, and Taliba Foster in our laboratory; Lynda Lanning of Pathology Associates International, Frederick, Maryland; Scott Perkins and Neil Lipman, Research Animal Resource Center, Cornell University Medical College; Manish Parikh, Division of Cardiology, Cornell University Medical College; and Monnie McGee-Harper, Rockefeller University Hospital GCRC Grant (RR00102).
Appendix: Discussion
Dr. Andrew S. Wechsler (Richmond, Va.). This work demonstrates the efficacy of Ad-based gene transfer as a means to achieve a potentially clinically applicable treatment strategy. It far transcends studies that have focused solely on demonstrating the ability to transfer genetic material to the myocardium. Locally enhanced expression of VEGF in the vicinity of ischemic myocardium improved collateral vessel density, regional blood flow, and myocardial function under stress conditions. The induction of new vessel growth by VEGF or its genetic progenitors is so promising that the Food and Drug Administration has recently approved a clinical trial proposed by Isner and his colleagues at the New England Medical Center. In their study, naked DNA coding for VEGF is to be infused into ischemic extremities unsuitable for surgical revascularization.
I have several questions for the authors:
Why did you select the 121 VEGF?
You noted minimal inflammation. Other investigators have consistently observed worrisome degrees of inflammation associated with Ad gene transfer. Why do you think inflammation was so minimal?
Why did you choose an Ad vector over naked DNA or plasmid-based gene transfer?
Have you performed experiments to determine the duration of gene expression or demonstrated VEGF within the myocardium?
Do you believe you have accelerated time-dependent collateral development or that you have induced a qualitative generation of neocollaterals?
Finally, do you think intravascular administration via the left anterior descending coronary artery would have had a comparable effect, inasmuch as this would allow a more convenient use of this technique if it were to become clinically applicable?
Dr. Mack. We chose the 121 isoform as opposed to the other isoforms because the 121 isoform is known to bind to just one of the VEGF receptors, unlike the other isoforms. Certain inflammatory cells such as monocytes and some carcinoma cell lines are known to express both or one of the VEGF receptors. We thought that the VEGF121 isoform would provide us with the most specificity for endothelial cells in that it binds to the Flk-1 receptor and not to the Flt-1 receptor.
In addition, VEGF121 does not have as much affinity for heparin as does VEGF165 and would diffuse more into the ischemic milieu and therefore potentially enhance any therapeutic effect.
Concerning inflammation, contamination by replication-competent viral particles to a large extent determines the degree of inflammation when Ad vectors are used. The vector that we used was free of replication-competent particles up to 108 pfu. As Ad vector technology advances, the degree of inflammation potentially can become less and less.
Disadvantages of the Ad vector for many of the inherited diseases, in that it expresses the transgene for only a limited duration, is one of the main reasons why we chose an Ad for this application. It was our hypothesis that a localized, yet transient degree of expression would be just enough to induce a neovascular response. Any prolonged degree of expression raises the concern of disorganized angiogenesis, inappropriate angiogenesis at other sites, such as the retina, occult tumors, and arthritic joints. The fact that the first-generation Ad vectors are known to express the transgene only transiently is a true advantage for this application,.
Regarding the duration of expression, in this porcine model we have done only very preliminary studies looking at gene expression and quantifying gene expression. Prior work in our laboratory done by Christopher Magovern has demonstrated that an Ad vector expressing VEGF165, expression can be as long as 7 to 14 days. Although we have not studied the duration of expression to date in the porcine model, we are planning to do that study and validate the fact that expression is transient.
Dr. Wechsler inquired about the mechanism contributing to our results, whether it is an increase in the endogenous response or a new generation of blood vessels. It is probably a combination of several things. The endogenous response probably is enhanced to some degree; however, other investigators, using VEGF and other growth factors and certain mitotic assays like bromodeoxyuridine and proliferating cell nuclear antigen, have demonstrated that a new generation of blood vessels develops. Therefore the collateral vessel response contributing to improved blood flow is likely a multifactorial process.
As far as the route of administration through the left anterior descending coronary artery, it was our purpose to directly inject the vector into the myocardium to avoid any systemic toxicity. Catheter-related devices that allow injection into the coronary vessel raise the concern of a systemic administration.
Dr. Margaret D. Allen (Seattle, Wash.). Did antibodies to Ad develop?
Dr. Mack. We looked at neutralizing antibodies at the time of Ameroid constrictor placement, the time of vector administration, and when the animals were put to death. Neutralizing antibodies to the Ad were observed at the time of sacrifice at a very low level. This has to be taken in context with what has been published for the most part in small animals, where the same dose of vector is given in an animal that is perhaps a thousand times smaller. The degree of neutralization in these experiments was relatively minimal.
Dr. Allen. That would be interesting, because it might affect retreatment in the future, for instance, for an ischemic patient who might need more than one treatment. It would be interesting to know whether the intramyocardial injection perhaps is less likely to induce an antibody response than an intravenous injection.
Dr. Mack. That is a very insightful point. Perhaps the route of administration does play a role here. As far as being able to administer the vector twice, there are 49 different Ad serotypes, and studies have been published whereby just altering the serotype can circumvent neutralizing immunity. This does not escape the cytotoxic T-cell response that clears the virus. However, clearly a repeat administration using an alternate serotype or immunomodulation can be achieved.
Mr. Reida M. El Oakley (London, England). I agree with Dr. Wechsler that your data would be more informative if you included two more control groups, one with the plasmid only and one with saline solution or nothing altogether.
Dr. Mack. We actually have in progress a treatment group in which the animals receive no injection at the second operation. Preliminarily, there appears to be no difference between that group and our AdNull-treated controls.
Footnotes
*R.G.C. and T.K.R. contributed equally as senior investigators in this study. 12/6/86191
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 M. H. Kown, T. Suzuki, M. L. Koransky, K. Penta, G. Sakamoto, C. L. Jahncke, A. J. Carter, T. Quertermous, and R. C. Robbins Comparison of developmental endothelial locus-1 angiogenic factor with vascular endothelial growth factor in a porcine model of cardiac ischemia Ann. Thorac. Surg., October 1, 2003; 76(4): 1246 - 1251. [Abstract] [Full Text] [PDF]
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 V. Chhokar and A. L. Tucker Angiogenesis: Basic Mechanisms and Clinical Applications Seminars in Cardiothoracic and Vascular Anesthesia, September 1, 2003; 7(3): 253 - 280. [Abstract] [PDF]
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G. Sakaguchi, Y. Sakakibara, K. Tambara, F. Lu, G. Premaratne, K. Nishimura, and M. Komeda A pig model of chronic heart failure by intracoronary embolization with gelatin sponge Ann. Thorac. Surg., June 1, 2003; 75(6): 1942 - 1947. [Abstract] [Full Text] [PDF]
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 G. C. Hughes, M. J. Post, M. Simons, and B. H. Annex Translational Physiology: Porcine models of human coronary artery disease: implications for preclinical trials of therapeutic angiogenesis J Appl Physiol, May 1, 2003; 94(5): 1689 - 1701. [Abstract] [Full Text] [PDF]
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T. Funatsu, Y. Sawa, S. Ohtake, T. Takahashi, G. Matsumiya, N. Matsuura, T. Nakamura, and H. Matsuda Therapeutic angiogenesis in the ischemic canine heart induced by myocardial injection of naked complementary DNA plasmid encoding hepatocyte growth factor J. Thorac. Cardiovasc. Surg., December 1, 2002; 124(6): 1099 - 1105. [Abstract] [Full Text]
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 B. S. Zuckerbraun and E. Tzeng Vascular Gene Therapy: A Reality of the 21st Century Arch Surg, July 1, 2002; 137(7): 854 - 861. [Abstract] [Full Text] [PDF]
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E. Leotta, G. Patejunas, G. Murphy, J. Szokol, L. McGregor, J. Carbray, A. Hamawy, D. Winchester, N. Hackett, R. Crystal, et al. Gene therapy with adenovirus-mediated myocardial transfer of vascular endothelial growth factor 121 improves cardiac performance in a pacing model of congestive heart failure J. Thorac. Cardiovasc. Surg., June 1, 2002; 123(6): 1101 - 1113. [Abstract] [Full Text] [PDF]
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C. L. Grines, M. W. Watkins, G. Helmer, W. Penny, J. Brinker, J. D. Marmur, A. West, J. J. Rade, P. Marrott, H. K. Hammond, et al. Angiogenic Gene Therapy (AGENT) Trial in Patients With Stable Angina Pectoris Circulation, March 19, 2002; 105(11): 1291 - 1297. [Abstract] [Full Text] [PDF]
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M.-H. Liu, H. Jin, H. S. Floten, Z. Ren, A. P.C. Yim, and G.-W. He Vascular endothelial growth factor-mediated, endothelium-dependent relaxation in human internal mammary artery Ann. Thorac. Surg., March 1, 2002; 73(3): 819 - 824. [Abstract] [Full Text] [PDF]
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 S. B. Freedman and J. M. Isner Therapeutic Angiogenesis for Coronary Artery Disease Ann Intern Med, January 1, 2002; 136(1): 54 - 71. [Abstract] [Full Text] [PDF]
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 R. Khurana, J. F. Martin, and I. Zachary Gene Therapy for Cardiovascular Disease: A Case for Cautious Optimism Hypertension, November 1, 2001; 38(5): 1210 - 1216. [Abstract] [Full Text] [PDF]
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J. M. Isner, P. R. Vale, J. F. Symes, and D. W. Losordo Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subjects Circ. Res., August 31, 2001; 89(5): 389 - 400. [Abstract] [Full Text] [PDF]
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Y. Lu, J. Shansky, M. Del Tatto, P. Ferland, X. Wang, and H. Vandenburgh Recombinant Vascular Endothelial Growth Factor Secreted From Tissue-Engineered Bioartificial Muscles Promotes Localized Angiogenesis Circulation, July 31, 2001; 104(5): 594 - 599. [Abstract] [Full Text] [PDF]
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 S. E. Epstein, S. Fuchs, Y. F. Zhou, R. Baffour, and R. Kornowski Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards Cardiovasc Res, February 16, 2001; 49(3): 532 - 542. [Abstract] [Full Text] [PDF]
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V. J. Dzau, M. J. Mann, A. Ehsan, and D. P. Griese Gene therapy and genomic strategies for cardiovascular surgery: The emerging field of surgiomics J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 0206 - 216. [Full Text] [PDF]
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H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, December 5, 2000; 97(25): 13801 - 13806. [Abstract] [Full Text] [PDF]
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K. A. Vincent, K.-G. Shyu, Y. Luo, M. Magner, R. A. Tio, C. Jiang, M. A. Goldberg, G. Y. Akita, R. J. Gregory, and J. M. Isner Angiogenesis Is Induced in a Rabbit Model of Hindlimb Ischemia by Naked DNA Encoding an HIF-1{alpha}/VP16 Hybrid Transcription Factor Circulation, October 31, 2000; 102(18): 2255 - 2261. [Abstract] [Full Text] [PDF]
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F. M. Belgore, A. D. Blann, G. Y. H. Lip, T. D. Henry, B. H. Annex, and T. F. Zioncheck sFlt-1, a Potential Antagonist for Exogenous VEGF Response Circulation, October 10, 2000; 102(15): e108 - e109. [Full Text] [PDF]
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E. Tanaka, N. Hattan, K. Ando, H. Ueno, Y. Sugio, M. U. Mohammed, S. A. Voltchikhina, and H. Mori Amelioration of microvascular myocardial ischemia by gene transfer of vascular endothelial growth factor in rabbits J. Thorac. Cardiovasc. Surg., October 1, 2000; 120(4): 720 - 728. [Abstract] [Full Text] [PDF]
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R. J. Lee, M. L. Springer, W. E. Blanco-Bose, R. Shaw, P. C. Ursell, and H. M. Blau VEGF Gene Delivery to Myocardium : Deleterious Effects of Unregulated Expression Circulation, August 22, 2000; 102(8): 898 - 901. [Abstract] [Full Text] [PDF]
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 I. Zachary, A. Mathur, S. Yla-Herttuala, and J. Martin Vascular Protection : A Novel Nonangiogenic Cardiovascular Role for Vascular Endothelial Growth Factor Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1512 - 1520. [Abstract] [Full Text] [PDF]
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M. Kawasuji, H. Nagamine, M. Ikeda, N. Sakakibara, H. Takemura, S. Fujii, and Y. Watanabe Therapeutic angiogenesis with intramyocardial administration of basic fibroblast growth factor Ann. Thorac. Surg., April 1, 2000; 69(4): 1155 - 1161. [Abstract] [Full Text] [PDF]
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R. Kornowski, M. B. Leon, S. Fuchs, Y. Vodovotz, M. A. Flynn, D. A. Gordon, A. Pierre, I. Kovesdi, J. A. Keiser, and S. E. Epstein Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy: Results in normal and ischemic porcine models J. Am. Coll. Cardiol., March 15, 2000; 35(4): 1031 - 1039. [Abstract] [Full Text] [PDF]
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R. Kornowski, S. Fuchs, M. B. Leon, and S. E. Epstein Delivery Strategies to Achieve Therapeutic Myocardial Angiogenesis Circulation, February 1, 2000; 101(4): 454 - 458. [Abstract] [Full Text] [PDF]
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R. C. Hendel, T. D. Henry, K. Rocha-Singh, J. M. Isner, D. J. Kereiakes, F. J. Giordano, M. Simons, and R. O. Bonow Effect of Intracoronary Recombinant Human Vascular Endothelial Growth Factor on Myocardial Perfusion : Evidence for a Dose-Dependent Effect Circulation, January 18, 2000; 101(2): 118 - 121. [Abstract] [Full Text] [PDF]
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 M. Donovan, M. Lin, P Wiegn, T Ringstedt, R Kraemer, R Hahn, S Wang, C. Ibanez, S Rafii, and B. Hempstead Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization Development, January 11, 2000; 127(21): 4531 - 4540. [Abstract] [PDF]
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L. Y. Lee, S. R. Patel, N. R. Hackett, C. A. Mack, D. R. Polce, T. El-Sawy, R. Hachamovitch, P. Zanzonico, T. A. Sanborn, M. Parikh, et al. Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121 Ann. Thorac. Surg., January 1, 2000; 69(1): 14 - 23. [Abstract] [Full Text] [PDF]
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J. F. Symes, D. W. Losordo, P. R. Vale, K. G. Lathi, D. D. Esakof, M. Mayskiy, and J. M. Isner Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease Ann. Thorac. Surg., September 1, 1999; 68(3): 830 - 836. [Abstract] [Full Text] [PDF]
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T. K. Rosengart, L. Y. Lee, S. R. Patel, T. A. Sanborn, M. Parikh, G. W. Bergman, R. Hachamovitch, M. Szulc, P. D. Kligfield, P. M. Okin, et al. Angiogenesis Gene Therapy : Phase I Assessment of Direct Intramyocardial Administration of an Adenovirus Vector Expressing VEGF121 cDNA to Individuals With Clinically Significant Severe Coronary Artery Disease Circulation, August 3, 1999; 100(5): 468 - 474. [Abstract] [Full Text] [PDF]
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 B.-G. Harvey, N. R. Hackett, T. El-Sawy, T. K. Rosengart, E. A. Hirschowitz, M. D. Lieberman, M. L. Lesser, and R. G. Crystal Variability of Human Systemic Humoral Immune Responses to Adenovirus Gene Transfer Vectors Administered to Different Organs J. Virol., August 1, 1999; 73(8): 6729 - 6742. [Abstract] [Full Text] [PDF]
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L. Poliakova, I. Kovesdi, X. Wang, M. C. Capogrossi, and M. Talan VASCULAR PERMEABILITY EFFECT OF ADENOVIRUS-MEDIATED VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSFER TO THE RABBIT AND RAT SKELETAL MUSCLE J. Thorac. Cardiovasc. Surg., August 1, 1999; 118(2): 339 - 347. [Abstract] [Full Text] [PDF]
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O. Reuthebuch, T. Podzuweit, S. Thomas, K. Binz, M. Roth, W.-P. Klovekorn, and E. P. Bauer Transmyocardial laser revascularisation has no beneficial effect on high energy phosphates and lactate content during acute myocardial ischaemia in pigs Eur J Cardiothorac Surg, August 1, 1999; 16(2): 144 - 149. [Abstract] [Full Text] [PDF]
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P. R. Vale, D. W. Losordo, T. Tkebuchava, D. Chen, C. E. Milliken, and J. M. Isner Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping J. Am. Coll. Cardiol., July 1, 1999; 34(1): 246 - 254. [Abstract] [Full Text] [PDF]
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L. Y. Lee, X. Zhou, D. R. Polce, T. El-Sawy, S. R. Patel, G. D. Thakker, K. Narumi, R. G. Crystal, and T. K. Rosengart EXOGENOUS CONTROL OF CARDIAC GENE THERAPY: EVIDENCE OF REGULATED MYOCARDIAL TRANSGENE EXPRESSION AFTER ADENOVIRUS AND ADENO-ASSOCIATED VIRUS TRANSFER OF EXPRESSION CASSETTES CONTAINING CORTICOSTEROID RESPONSE ELEMENT PROMOTERS J. Thorac. Cardiovasc. Surg., July 1, 1999; 118(1): 26 - 25. [Abstract] [Full Text] [PDF]
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 A. Basile-Borgia, J. H Abel, and H. Mahloogi Molecular advances in cardiac and cardiovascular disease Perfusion, March 1, 1999; 14(2): 89 - 99. [Abstract] [PDF]
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D. W. Losordo, P. R. Vale, J. F. Symes, C. H. Dunnington, D. D. Esakof, M. Maysky, A. B. Ashare, K. Lathi, and J. M. Isner Gene Therapy for Myocardial Angiogenesis : Initial Clinical Results With Direct Myocardial Injection of phVEGF165 as Sole Therapy for Myocardial Ischemia Circulation, December 22, 1998; 98(25): 2800 - 2804. [Abstract] [Full Text] [PDF]
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E. J. Topol and P. W. Serruys Frontiers in Interventional Cardiology Circulation, October 27, 1998; 98(17): 1802 - 1820. [Full Text] [PDF]
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H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, December 5, 2000; 97(25): 13801 - 13806. [Abstract] [Full Text] [PDF]
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 G. B. Dalshaug, T. D. Scholz, O. M. Smith, K. A. Bedell, C. A. Caldarone, and J. L. Segar Effects of gestational age on myocardial blood flow and coronary flow reserve in pressure-loaded ovine fetal hearts Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1359 - H1369. [Abstract] [Full Text] [PDF]
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 S. C. FRANCIS, M. K. RAIZADA, A. A. MANGI, L. G. MELO, V. J. DZAU, P. R. VALE, J. M. ISNER, D. W. LOSORDO, J. CHAO, M. J. KATOVICH, et al. Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb? Physiol Genomics, December 21, 2001; 7(2): 79 - 94. [Abstract] [Full Text] [PDF]
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H. Iwaguro, J.-i. Yamaguchi, C. Kalka, S. Murasawa, H. Masuda, S.-i. Hayashi, M. Silver, T. Li, J. M. Isner, and T. Asahara Endothelial Progenitor Cell Vascular Endothelial Growth Factor Gene Transfer for Vascular Regeneration Circulation, February 12, 2002; 105(6): 732 - 738. [Abstract] [Full Text] [PDF]
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